Abstract:
A nucleic acid amplification apparatus for amplifying a target nucleic acid derived from living organism, comprising: a measurement unit for amplifying the target nucleic acid in a measuring sample prepared from the living organism, and measuring a product of the amplification of the target nucleic acid; a measurement value obtaining unit for obtaining a measurement value related to an amount of the product of the amplification; and a judging unit for judging whether amplification inhibition of the target nucleic acid occur or not based on a first measurement value and a second measurement value, the first measurement value obtained from a first measurement sample and the second measurement value obtained from a second measurement sample having a difference dilution ratio from the first measurement sample.

Description:
FIELD OF THE INVENTION 
     The present invention relates to a nucleic acid amplification apparatus and method and more particularly, to a nucleic acid amplification and method capable of amplifying and measuring a target nucleic acid in a sample derived from living organism. 
     BACKGROUND 
     In recent years, gene testing has been becoming rapidly widely used in the clinical diagnosis field. With gene testing, nucleic acids and chromosomes are analyzed to allow for examination of presence or absence of variations and nuclear forms associated with hereditary disorders for clinical purposes. As one example of gene testing, diagnosis of cancer cell metastasis to lymph nodes is mentioned. Cancer cells leave primary tumor and spread by metastasis all over patient&#39;s body via blood vessels and lymph ducts. At operation of a cancer, lesions should be removed as much surely as possible, and therefore, metastasis should be detected accurately and appropriate treatments be provided depending on the degree of metastasis. In this sense, diagnosis of cancer cell metastasis to lymph nodes made during the operation has an extremely important meaning. As one of methods of diagnosis of cancer cell metastasis to lymph nodes, such a method is known that nucleic acid of a protein, that is not expressed in normal cells or expression level thereof is low but is expressed a great deal in cancer cells, is detected as a target nucleic acid. Thanks to advancement of gene analysis technology made in these years, it is now possible to perform cancer diagnosis effectively by detecting a target nucleic acid contained in the lymph node tissue resected from the living organism through amplification. 
     As mentioned above, when it is desired to make judgment of cancer cell metastasis to lymph nodes by amplification of target nucleic acid, amplification of target nucleic acids in a measurement sample is performed using a measurement sample which is prepared in such that lymph nodes are homogenized and target nucleic acids are extracted into a solution and purified. However, with this method, there is such a drawback that purification of the target nucleic acid needs considerable time, it takes longer time before results of the judgment by amplification of the target nucleic acid are made available, and it is difficult to perform promptly diagnosis of cancer cell metastasis by amplification of the target nucleic acid. At diagnosis of cancer cell metastasis to lymph nodes during the operation, treatment strategy in the operation is determined according to results of judgment of metastasis of cancer cells, and therefore, quick judgment of metastasis is so important. 
     From viewpoints mentioned above, if a solution in which lymph nodes are homogenized or supernatant of this solution is used as the measurement sample without executing extraction and purification of the nucleic acid at the time of preparation of measurement sample, it is possible to perform measurements of the target nucleic acid in prompt fashion. However, when the target nucleic acid is amplified using such a measurement sample, there is such a problem that, compared to a case where amplification of nucleic acids is made using a measurement sample prepared by purification of the nucleic acid, the amount of inhibitory substances that prevent amplification of the target nucleic acid derived from lymph nodes increases and accurate measurement results can not be thus obtained. 
     In order to overcome this problem, conventionally, such a method is known that amplification of a target nucleic acid is estimated using a nucleic acid probe which hybridizes to the target nucleic acid (see, for example, Japanese Patent Application Laid-Open No. 2004-203). According to the method disclosed in Japanese Patent Application Laid-Open No. 2004-203, a nucleic acid (internal standard nucleic acid) in which base sequence of the target nucleic acid is mutated in part is added to a measuring system at known concentration, and at the same time, a target nucleic acid probe that hybridizes specifically to the target nucleic acid and an internal standard nucleic acid probe that hybridizes specifically to the internal standard nucleic acid are added to the measuring system, the target nucleic acid and the internal standard nucleic acid are then measured at one time using PCR method, and the target nucleic acid is measured from an amount of addition of the internal standard nucleic acid. 
     However, with the method for measuring the target nucleic acid disclosed in above-mentioned Japanese Patent Application Laid-Open No. 2004-203, the internal standard nucleic acid does not necessarily exhibit the same reactivity as the target nucleic acid does, and therefore, a problem arises that accurate measurement of the target nucleic acid is difficult. 
     SUMMARY 
     The scope of the present invention is defined solely by the appended claims, and is not affected by any degree by the statements within this summary. 
     A nucleic acid amplification apparatus according to a first aspect of the present invention is a nucleic acid amplification apparatus for amplifying a target nucleic acid derived from living organism, comprising: a measurement unit for amplifying the target nucleic acid in a measuring sample prepared from the living organism, and measuring a product of the amplification of the target nucleic acid; a measurement value obtaining unit for obtaining a measurement value related to an amount of the product of the amplification; and a judging unit for judging whether amplification inhibition of the target nucleic acid occur or not based on a first measurement value and a second measurement value, the first measurement value obtained from a first measurement sample and the second measurement value obtained from a second measurement sample having a difference dilution ratio from the first measurement sample. 
     A nucleic acid amplification method according to a second aspect of the present invention is a nucleic acid amplification method for amplifying a target nucleic acid derived from living organism, comprising steps of: amplifying the target nucleic acid in a first measuring sample prepared from the living organism; amplifying the target nucleic acid in a second measuring sample prepared from the living organism and had a difference dilution ratio from the first measurement sample; measuring a first product of the amplification of the target nucleic acid of the first measurement sample; measuring a second product of the amplification of the target nucleic acid of the second measurement sample; obtaining a first measurement value related to an amount of the first product of the amplification; obtaining a second measurement value related to an amount of the second product of the amplification; and judging whether amplification inhibition of the target nucleic acid occur or not based on the first measurement value and the second measurement value. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective illustration showing whole composition of a gene amplification and analysis system by one embodiment according to the present invention. 
         FIG. 2  is a perspective illustration showing whole composition of a gene amplification measuring apparatus of the gene amplification and analysis system by one embodiment shown in  FIG. 1 . 
         FIG. 3  is a schematic plan view of  FIG. 2 . 
         FIG. 4  is a drawing showing data browser screen displayed on the display unit of personal computer composing the gene amplification and analysis system by one embodiment shown in  FIG. 1 . 
         FIG. 5  is a drawing showing relationship between measurement results of sample specimen, measurement results of dilution sample, and existence or nonexistence of target gene. 
         FIG. 6  is a graph showing relationship between dilution rate and concentration of sample specimen that caused amplification inhibition. 
         FIG. 7  is a drawing showing workload list screen displayed on the display unit of the personal computer composing the gene amplification and analysis system by one embodiment shown in  FIG. 1 . 
         FIG. 8  is a drawing showing analytical curve display screen displayed on the display unit of the personal computer composing the gene amplification and analysis system by one embodiment shown in  FIG. 1 . 
         FIG. 9  is a schematic plan view showing variation of the gene amplification measuring apparatus of the gene amplification and analysis system by one embodiment shown in  FIG. 1 . 
         FIG. 10  is a flowchart showing amplification inhibition judgment flow of CPU of the personal computer of the gene amplification and analysis system by one embodiment shown in  FIG. 1 .  FIG. 1  is a plan view showing whole composition of an immune analysis apparatus equipped with a pipette chip supplying apparatus by one embodiment according to the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Now, embodiments of the present invention will be explained hereinafter referring to the drawings. 
     A gene amplification and analysis system  1  according to the present embodiment is a system supporting cancer metastasis diagnosis for resected tissue (lymph node) at operation of cancer, wherein target gene (mRNA) originated from cancer existing in the resected tissue is amplified by LAMP (Loop-Medicated Isothermal Amplification, Eiken Chemical Co., Ltd.) method, and white turbidity due to magnesium pyrophosphate, which is generated by amplification, is measured by turbidimetry to know whether or not the target gene is present at a predetermined level or more. Meanwhile, details of the LAMP method are disclosed in U.S. Pat. No. 6,410,278. 
     The gene amplification and analysis system  1  of the present embodiment comprises, as shown in  FIG. 1 , a gene amplification measuring apparatus  101  and a personal computer (PC)  102  connected so as to allow communication with the gene amplification measuring apparatus  101  in wired or wireless fashion. 
     First, referring to  FIGS. 1 to 3 , details of the gene amplification measuring apparatus  101  will be explained. The gene amplification measuring apparatus  101  includes, as shown in  FIGS. 2 and 3 , a dispensing mechanism  10 , a sample specimen setting unit  20 , a chip setting unit  30 , a chip disposal unit  40 , and a reaction detection unit  50  comprising five reaction detection blocks  50   a , and a transfer unit  60  for transferring the dispensing mechanism  10  in X-axis direction and Y-axis direction. 
     Further, the dispensing mechanism  10  includes, as shown in  FIGS. 2 and 3 , an arm portion  11  which is moved in X-axis direction and Y-axis direction (horizontal direction) by the transfer unit  60 , and a double (two) syringe unit  12  each capable of moving independently in Z-axis direction (vertical direction) with regard to the arm portion  11 . 
     Further, as shown in  FIGS. 2 and 3 , to the sample specimen setting unit  20  are provided, sequentially from the front of the apparatus, ten sample container setting holes  21   a . through  21   j , one enzyme reagent container setting hole  21   k . and one primer reagent container setting hole  21 I. Further, ten sample container setting holes  21   a . through  21   j . are provided being arranged in five lines and two rows. And, sample container setting holes  21   c . and  21   d , sample container setting holes  21   e  and  21 f, sample container setting holes  21   g . and  21   h , sample container setting holes  21   i . and  21   j . are provided to, sequentially from the back of the apparatus, sample setting position  1 , sample setting position  2 , sample setting position  3 , and sample setting position  4 . 
     Besides, according to the present embodiment, to the sample container setting holes  21   c ,  21   e ,  21   g . and  21   i . at the left of the front, is set a sample container  22  in which solubilization extraction liquid (sample specimen), produced in advance by treatment (homogenization, filtering or the like) of a resected living tissue (lymph node), is accommodated, and to the sample container setting holes  21   d ,  21   f ,  21   h . and  21   j . at the right of the front, is set a sample container  23  in which each of dilution samples (ten-fold dilution) of above-mentioned sample specimens are accommodated. Specifically, a dilution sample corresponding to a sample specimen accommodated in the sample container  22  which is to be set in the sample container setting hole  21   c . is accommodated to a sample container  23  of the sample container setting hole  21   d . Further, a dilution sample corresponding to a sample specimen accommodated in the sample container  22  which is to be set in the sample container setting hole  21   e . is accommodated to a sample container  23  of the sample container setting hole  21   f , a dilution sample corresponding to a sample specimen accommodated in the sample container  22  which is to be set in the sample container setting hole  21   g . is accommodated to a sample container  23  of the sample container setting hole  21   h , a dilution sample corresponding to a sample specimen accommodated in the sample container  22  which is to be set in the sample container setting hole  21   i . is accommodated to a sample container  23  of the sample container setting hole  21   j . In other words, two samples (sample specimen, dilution sample) are produced from one living tissue. In the meantime, in the present embodiment, unpurified samples (target nucleic acid is not purified) are used as the sample specimen and the dilution sample. 
     Further, a container  24 , in which a positive control for confirming that a gene to be amplified is amplified normally is accommodated, is placed to the sample container setting hole  21   a . and at the same time, a container  25 , in which a negative control for confirming that a gene not to be amplified is not amplified normally is accommodated, is set in a sample container setting hole  21   b.    
     To an enzyme reagent container setting hole  21   k . and to a primer reagent container setting hole  211  are set an enzyme reagent container  26  accommodating DNA polymerase and reverse transfer enzyme as an enzyme reagent and a primer reagent container  27  accommodating primer reagent of CK19. are set, respectively. 
     Further, as shown in  FIG. 3 , two racks  32  each having an accommodation hole  32   a . capable of accommodating  36  pieces of pipette chips  31  are inserted detachably into the chip setting unit  30 . Two removal buttons  33  are provided to the chip setting unit  30 . By pressing the removal button  33 , racks  32  are put into removable state. 
     As shown in  FIG. 3 , two chip disposal holes  40   a . for disposal of used pipette chip  31  are provided to the chip disposal unit  40 . Besides, a groove portion  40   b . having width narrower than the chip disposal hole  40   a . is provided to be continued to the chip disposal hole  40   a.    
     Further, as shown in  FIGS. 2 and 3 , each of reaction detection blocks  50   a . of the reaction detection unit  50  comprises a reaction unit  51 , two turbidity detection units  52  and a cover closing mechanism  53  (see  FIG. 3 ). As shown in  FIG. 3 , to the reaction unit  51  provided to each of reaction detection blocks  50   a . is provided two detection cell setting holes  51   a . for setting a detection cell  54 . Each of the reaction detection blocks  50   a  is disposed to, sequentially from the back of the apparatus, cell setting position  1 , cell setting position  2 , cell setting position  3 , cell setting position  4 , and cell setting position  5 . 
     Further, the turbidity detection units  52  comprise, as shown in  FIG. 3 , an LED light source  52   a . comprising blue LED having 465. nm wavelength which is mounted to a substrate  55   a  disposed at one side face of the reaction unit  51 , and a photodiode light receiving unit  52   b . which is mounted to a substrate  55   b . disposed to other side face of the reaction unit  51 . One set of turbidity detection units  52  each comprising one LED light source  52   a . and one photodiode light receiving unit  52   b . is disposed by two sets to each of reaction detection blocks  50   a . Therefore, the turbidity detection unit  52  comprising a total ten sets of LED light sources  52   a . and photodiode light receiving units  52   b . is disposed to five reaction detection blocks  50   a . The LED light source  52   a . and the photodiode light receiving unit  52   b . corresponding thereto are disposed so that a light approximately 1. mm in the diameter is irradiated from the LED light source  52   a . to lower part of the detection cell  54  and this light could be received by the photodiode light receiving unit  52   b . With the use of intensity of the light received by the photodiode light receiving unit  52   b , presence or absence of the detection cell  54  can be detected and turbidity of the liquid accommodated inside of the cell unit  54   a . of the detection cell  54  can be monitored by a display unit  102   c . of the personal computer  102 , which will be described later. Specifically, when the detection cell  54  is set in the detection cell setting hole  51   a , since the detection cell  54  is being disposed between the LED light source  52   a . and photodiode light receiving unit  52   b , a light received by the photodiode light receiving unit  52   b . becomes weaker than the case where the detection cell  54  is not set. It is now possible to detect that the detection cell  54  is being set. 
     Further, the detection cell  54  has two cell units  54   a . for accommodating the sample specimen and the dilution sample, and two cover units  54   b . for covering two cell units  54   a.    
     Further, the transfer unit  60  includes, as shown in  FIGS. 2 and 3 , a direct operation guide  61  and ball screw  62  for transferring the dispensing mechanism  10  in Y-axis direction, a stepping motor  63  for driving the ball screw  62 , a direct operation guide  64  and a ball screw  65  for transferring the dispensing mechanism  10  in X-axis direction, a stepping motor  66  for driving the ball screw  65 . Meanwhile, transfers of the dispensing mechanism  10  in X-axis direction and in Y-axis direction are performed by turning the ball screw  62  and  65  by the stepping motor  63  and  66 , respectively. 
     The personal computer  102  includes, as shown in  FIG. 1 , a keyboard  102   a . and a mouse  102   b . serving as the input device, the display unit  102   c . comprising a monitor, and a CPU  102   d . for analyzing measurement results of the sample specimen and the dilution sample. Next, referring to  FIG. 1  and  FIGS. 4 to 8 , details of screen layout of the display unit  102   c . of the personal computer  102  will be explained. 
     The display unit  102   c . is provided to display a screen (data browser screen) for displaying measurement results of the sample specimen being analyzed by the CPU  102   d , a screen (workload list screen) for giving measurement instructions such as registration of sample ID using the keyboard  102   a . and the mouse  102   b , and a screen (analytical curve display screen) for displaying the analytical curve, or the like. 
     On the data browser screen, as shown in  FIG. 4 , a tool bar  111  which displays buttons for executing various functions such as help function, a sample information display unit  112  for displaying various information of the sample specimen, and a measurement results display unit  113  for indicating measurement results of the sample specimen which are displayed on the sample information display unit  112 , are displayed. 
     Further, on the sample information display unit  112  are provided a batch number display column  112   a , a sample position display column  112   b , a sample ID display column  112   c , a comment display column  112   d , a measurement date display column  112   e , and a measurement time display column  112   f . The batch number display column  112   a . shows what number of batch processing is taking place. Meanwhile, batch processing means that a plurality of sample specimens and dilution samples are processed collectively (in the present embodiment, a maximum of four sample specimens and a maximum of four dilution samples) In the batch number display column  112   a , a numeral showing the number of times of batch processing executed after power supplying plus “1” is shown (“5” in the screen). In the sample position display column  112   b , sample setting position where the sample specimen is being set is displayed (“4” in the screen). In the sample ID display column  112   c . and comment display column  112   d , comments for sample ID of the sample specimen entered in the workload list screen which will be described later (“Sample 02” on the screen) and for the sample specimen (dilution sample) (blank on the screen) are displayed, respectively. Further, in the measurement date display column  112   e . and measurement time display column  112   f , date of measurement of the sample specimen and the dilution sample (“2005/09/26” on the screen) and time (“12:53:06” on the screen) are displayed. 
     Further, on the measurement results display unit  113 , graph  113   a . showing relationship between turbidity of the sample specimen identified from above-mentioned batch number display column  112   a . and sample position display column  112   b . and time (min), amplification rising time display column  113   b , concentration measurement display column  113   c , and judgment result display column  113   d . are provided. In the meantime, according to the present embodiment, measurement results of the dilution sample (graph, amplification rising time, concentration measurement and judgment results) are set so that users other than the administrator are not permitted to take a look. As a result, it is possible to remove such a chance that users may consider measurement result of the dilution sample as measurement result of the sample specimen. 
     Further, in the amplification rising time display column  113   b , time corresponding to turbidity 0.1. on the vertical axis of the graph  113   a . (“10.7” on the screen) is displayed. 
     Further, according to the present embodiment, in the concentration measurement display column  113   c , concentration or range of concentration of the sample specimen calculated from the rising time (=10.7) (min) displayed on the amplification rising time display column  113   b . (“&lt;2.5E+02” on the screen) (copies/μl) is displayed. Specifically, concentration is calculated from amplification rising time (=10.7) based on the analytical curve (see  FIG. 8 ) that is a linear function of amplification rising time and concentration prepared by a calibrator measured in advance. When the concentration is not less than 2.5×10 2 . (copies/μl), concentration actually measured is displayed within the range of linearity assurance “2.5E+07. copies/μl”, and when it is less than 2.5×10 2 . (copies/μl), “&lt;2.5E+02” is displayed. When the range of linearity assurance “2.5E+07. copies/μl” is exceeded, “&gt;2.5E+07” is displayed. 
     According to the present embodiment, the judgment result display column  113   d . is provided to display the result of whether or not a target gene (mRNA) is present at a predetermined level or more in the sample specimen (positive “+”, negative “−”) based on the measurement result (concentration) of the sample specimen, and the measurement result (concentration) of dilution sample thereof. Further, the judgment result display column  113   d . is provided to display information about amplification inhibition of target gene (mRNA) in the sample specimen together with the result of whether or not the target gene is present at the predetermined level or more as mentioned above. Specifically, as shown in  FIG. 5 , when measurement result of the sample specimen (CK19) is not less than 2.5×10 2  (copies/μl), “(+)” showing positive is displayed. When measurement result of a sample specimen is less than 2.5×10 2  (copies)/μl and measurement result of the dilution sample (CK19-D) of the sample specimen is less than 2.5×10 2 . (copies)/μl, showing negative is displayed. When measurement result of the sample specimen (CK19) is less than 2.5×10 2 . (copies)/μl and measurement result of the dilution sample (CK19-D) of the sample specimen is not less than 2.5×10 2 . (copies)/μl, “(+) I” showing positive is displayed. Data browser screen shown in  FIG. 4  shows an example of a case where measurement result of the sample specimen (CK19) is less than 2.5×10 2 . (copies)/μl and measurement result of the dilution sample (CK19-D) of the sample specimen is not less than 2.5×10 2 . (copies)/μl. This is such a case where, as shown in  FIG. 6 , although measurement result taken using a dilution sample (dilution of a sample specimen) is positive, measurement result taken using the sample specimen is negative. Meanwhile, “I” in “(+) I” that is shown when measurement result of a sample specimen is less than 2.5×10 2  (copies)/μl and measurement result of a dilution sample of the sample specimen is not less than 2.5×10 2 . (copies)/μl, is a graph indicating that amplification inhibition occurred. 
     On the workload list screen, as shown in  FIG. 7 , a tool bar  121  on which buttons for executing various functions such as printing function are displayed, an order entry unit  122  for entering measurement order (measurement indication), an order list display unit  123  for displaying status of measurement order registration, a batch number display column  124 , a group selection column  125 , cell setting position display units  126   a  to  126   e , a sample setting position display unit  127 , and a measurement start button  128  are displayed. 
     Further, the order entry unit  122  is provided to execute entry of measurement order for sample setting positions  1  to  4 , and entry of measurement order for accuracy control sample (positive control, negative control) to be set in the sample container setting holes  21  and  21   b . (see  FIG. 3 ). To this order entry unit  122  are provided a sample ID entry column  122   a , a comment entry column  122   b . and an enter button  122   c.  Specifically, using the keyboard  102   a , sample ID is entered to the sample ID entry column  122   a . for sample specimen at sample setting positions  1  to  4 , and accuracy control sample of sample container setting holes  21   a . and  21   b . As for the sample ID, ID corresponding to negative control or positive control as well as ID corresponding to the sample specimen is entered. As for ID of a sample, for example, “Sample 01˜Sample 04” are used. As for sample ID of positive control, for example, “QC [CK19-PC]” is used. As for sample ID of negative control, for example, “QC [CK19-NC]” is used. When there is a comment(s), it is possible to enter the comment to the comment entry column  122   b . of the order entry unit  122 . When the enter button  122   c  is clicked by the mouse  102   b , the sample ID and comment(s) being entered are reflected to the order list display unit  123 . 
     The batch number display column  124  displays what number of batch processing is taking place in similar fashion as the batch number display column  112   a . of the sample information display unit  112  of data browser screen (see  FIG. 4 ). In the group selection column  125 , a group is selected from a pulldown menu  125   a . As for this group, for example, a group for measuring sample specimen, a group for measuring calibrator for obtaining an analytical curve, or the like is mentioned. The present embodiment shows an example of the case where the group for measuring a sample specimen (Sample) is selected. When this group (Sample) is selected, “O” is displayed at a place corresponding to CK19. on the order list display unit  123 . 
     Further, cell setting position display units  126   a . to  126   e  are provided to display set status of the detection cell  54  of each of reaction detection blocks  50   a . of the reaction detection unit  50 . As for set status of the detection cell  54 , when use is scheduled and the detection cell  54  is set in the detection cell setting hole  51   a , “G” (displayed in green) is displayed on the cell setting position display units  126   a . and  126   b , as shown in  FIG. 7 . When the detection cell  54  is not set in the detection cell setting hole  51   a . although use is scheduled, “NG” (displayed in red) is displayed at a predetermined place of the cell setting position display units  126   a . to  126   e . When setting of the detection cell  54  to the detection cell setting hole  51   a  is not required since use is not scheduled, a pattern (displayed in grey) showing the reaction unit  51  in a state, that there is no need for setting the detection cell  54  at a predetermined position of the cell setting position display units  126   a . to  126   e , is displayed. 
     Further, the sample setting position display unit  127  is provided to display set status of the sample container  22  for accommodating a sample specimen of the sample specimen setting unit  20  of the gene amplification measuring apparatus  101 , the sample container  23  for accommodating dilution sample, the container  24  for accommodating positive control, the container  25  for accommodating negative control, the enzyme reagent container  26 , and the primer reagent container  27 . The sample setting position display unit  127  has sample container display units  127   a . to  127   j . corresponding to ten sample container setting holes  21   a . to  21   j , an enzyme reagent container display unit  127   k  corresponding to the enzyme reagent container setting hole  21   k , and a primer reagent container display unit  127 I corresponding to the primer reagent container setting hole  21 I. Alphabets (“PC” on the screen) corresponding to sample ID (QC “CK19-PC”) displayed on the order list display unit  123  are displayed on the sample container display unit  127   a . Besides, alphabets (“NC” on the screen) corresponding to sample ID (QC “CK19-NC”) displayed on the order list display unit  123  are displayed on the sample container display unit  127   b.    
     Further, an alphabet (“S” showing sample on the screen) corresponding to sample ID displayed on the order list display unit  123  is displayed on the sample container display units  127   c ,  127   e ,  127   g . and  127   i . An alphabet (“D” showing dilution on the screen) showing a dilution sample is displayed on sample container display units  127   d ,  127   f ,  127   h . and  127   j . On the enzyme reagent container display unit  127   k . is displayed an alphabet (“E” on the screen) showing that the enzyme reagent container  26  is being set, and on the primer reagent container display unit  127 I is displayed an alphabet (“P” on the screen) showing that the primer reagent container  27  is placed. In the present embodiment, a screen showing that entry of measurement order for sample setting position  1  has been completed is shown. 
     The analytical curve display screen is, as shown in FIG.  8 , a screen for displaying an analytical curve prepared by measuring calibrator of three known concentrations (2.5×10 3  (copies/μl), 2.5×10 5 . (copies/μl), 2.5×10 7 . (copies/μl)), and three concentration points plotted against calibrator rising amplification time are represented by a straight line approximated by linear expression. 
     Next, referring to  FIGS. 1 to 4 ,  FIGS. 7 and 8 , operations of the gene amplification analysis system  1  according to the present embodiment will be explained. With the gene amplification analysis system  1  according to the present embodiment, as mentioned above, a target gene (mRNA) derived from a cancer which is present in a tissue resected at cancer operation is amplified using LAMP method, white turbidity due to magnesium pyrophosphate generated by amplification is measured to make judgment whether or not the target gene exists at the predetermined level or more. In the meantime, with LAMP method used in the present embodiment, although a treatment for extracting the target gene (mRNA) by treating the resected tissue is performed, no purification treatment is performed. 
     First, as shown in  FIG. 2  and  FIG. 3 , the sample containers  22 , in which is accommodated a solubilization extraction liquid (sample specimen) produced in advance by treatment (homogenization, filtering or the like) of the resected tissue, are set in the sample container setting holes  21   c ,  21   e ,  21   g . and  21   i . The sample containers  23  accommodating the dilution samples in which sample specimen to be accommodated in the sample container  22  is diluted ten-fold, are set in the sample container setting holes  21   d ,  21   f ,  21   h . and  21   j . The container  24  in which positive control is accommodated and the container  25  in which negative control is accommodated are set in the sample container setting hole  21   a . and  21   b , respectively (see  FIG. 3 ). The enzyme reagent container  26  in which enzyme reagent of CK19. is accommodated, and the primer reagent container  27  in which primer reagent of CK19. is accommodated are set in the enzyme reagent container setting hole  21   k . (see  FIG. 3 ) and the primer reagent container setting hole  21 I respectively. Further, two racks  32  each accommodating  36  pieces of disposable pipette chips  31  are mounted to the chip setting unit  30 . 
     Before starting measurements, measurement instructions such as registration of sample ID are given on the screen of the display unit  102   c . of the personal computer  102  (workload list screen (see  FIG. 7 ) ) using the keyboard  102   a . and the mouse  102   b . of the personal computer  102  shown in  FIG. 1 . 
     The user then clicks, using the mouse  102   b . (see  FIG. 1 ), the measurement start button  128  on the workload list screen shown in  FIG. 7 . With this manipulation, measurement operations of the gene amplification measuring apparatus  101  are started. 
     When operations of the gene amplification measuring apparatus  101  are started, first, the arm portion  11  of the dispensing mechanism  10  is moved from an initial position to the chip setting unit  30  by the transfer unit  60  shown in  FIG. 2 , and then, at the chip setting unit  30 , two syringe units  12  of the dispensing mechanism  10  are moved downwardly. By these operations, a front edge of nozzle part of each of two syringe units  12  is press fit into an upper opening of each of the two pipette chips  31  and therefore, the pipette chip  31  is automatically mounted to the front edge of nozzle part of each of two syringe units  12 . After two syringe units  12  are moved upwardly, the arm portion  11  of the dispensing mechanism  10  is moved in X-axis direction towards upper part of the primer reagent container  27  in which primer reagent of CK19. is accommodated. After one syringe unit  12  located at upper part of the primer reagent container  27  is moved downwardly and primer reagent is sucked, other syringe unit  12  is then moved upwardly. After that, the arm portion  11  of the dispensing mechanism  10  is moved in Y-axis direction by the transfer unit  60  so that other syringe unit  12  may be positioned at upper part of the same primer reagent container  27 . After other syringe unit  12  is moved downwardly and primer reagent is sucked from the same primer reagent container  27 , the other syringe unit  12  is moved upwardly. In this way, primer reagent of CK19. in the primer reagent container  27  is sucked by two pipette chips  31  mounted to the syringe unit  12 . 
     After the primer reagent is sucked and after two syringe units  12  are moved upwardly, the arm portion  11  of the dispensing mechanism  10  is moved by the transfer unit  60  above reaction detection block  50   a . which is positioned at the cell setting position  1  that is the deepest (back of apparatus front). When two syringe units  12  are moved downwardly at the reaction detection block  50   a . which is located at the deepest, two pipette chips  31  mounted to two syringe units  12  are inserted into two cell units  54   a . of the detection cell  54 , respectively. Following this, using the syringe unit  12 , the primer reagent of CK19. is discharged to two cell units  54   a , respectively. 
     After primer reagent is discharged and then two syringe units  12  are moved upwardly, the arm portion  11  of the dispensing mechanism  10  is moved by the transfer unit  60  in X-axis direction towards upper part of the chip disposal unit  40 . Then, disposal of the pipette chip  31  is performed at the chip disposal unit  40 . Specifically, after two syringe units  12  are moved downwardly, the pipette chip  31  is inserted into two chip disposal holes  40   a . (see  FIG. 3 ) of the chip disposal unit  40 . In this state, when the arm portion  11  of the dispensing mechanism  10  is moved in Y-axis direction by the transfer unit  60 , the pipette chip  31  is moved under the groove portion  40   b.  When two syringe units  12  are moved upwardly, flange portion on upper plane of the pipette chip  31  abuts lower plane at both sides of the groove portion  40   b , thereby receiving a downward force from the lower plane, and therefore, the pipette chip  31  is automatically disengaged from nozzle part of each of two syringe units  12 . By these operations, the pipette chip  31  is discarded to the chip disposal unit  40 . 
     Next, the arm portion  11  of the dispensing mechanism  10  is moved again by the transfer unit  60  to the chip setting unit  30 . Following this, at the chip setting unit  30 , two new pipette chips  31  are automatically mounted to the front edge of the nozzle part of each of two syringe units  12  by the same operations as mentioned above. The arm portion  11  of the dispensing mechanism  10  is moved in X-axis direction towards upper part of the enzyme reagent container  26  in which is accommodated enzyme reagent of CK19. After one syringe unit  12  located at upper part of the enzyme reagent container  26  is moved downwardly and enzyme reagent is being sucked, the one syringe unit  12  is moved upwardly. Following this, the arm portion  11  of the dispensing mechanism  10  is moved in Y-axis direction by the transfer unit  60  so that other syringe unit  12  may be positioned at upper part of the same enzyme reagent container  26 . After other syringe unit  12  is moved downwardly and enzyme reagent is being sucked from the same enzyme reagent container  26 , the other syringe unit  12  is moved upwardly. In this way, enzyme reagents in the enzyme reagent container  26  are sucked by two pipette chips  31  mounted to the syringe unit  12 . 
     After the arm portion  11  of the dispensing mechanism  10  is moved to upper part of the reaction detection block  50   a  located at the deepest by the transfer unit  60 , the enzyme reagent of CK19. is discharged to two cell units  54   a . of the detection cell  54 . After the enzyme reagent is discharged, and after the arm portion  11  of the dispensing mechanism  10  is moved above the chip disposal unit  40  by the transfer unit  60 , disposal of the pipette chip  31  is performed. 
     Next, the arm portion  11  of the dispensing mechanism  10  is moved again by the transfer unit  60  to the chip setting unit  30 , two new pipette chips  31  are automatically mounted to the front edge of the nozzle part of each of two syringe units  12 . The arm portion  11  of the dispensing mechanism  10  is moved in X-axis direction towards upper part of the sample container  22  and sample container  23  in which are accommodated the sample specimen and the dilution sample being set to the sample specimen setting unit  20 , and after that, the sample specimen and the dilution sample in the sample containers  22  and  23  are sucked at once by the same suction operations of primer reagent and enzyme reagent as mentioned above. After that, the arm portion  11  of the dispensing mechanism  10  is moved above the reaction detection block  50   a . located at the deepest by the transfer unit  60 , and then two syringe units  12  are moved downwardly, and the sample specimen and the dilution sample are discharged to two cell units  54   a . of the detection cell  54 , respectively. Meanwhile, when dispensing primer reagent, enzyme reagent and sample specimen (dilution sample), temperature of liquid in the detection cell  54  is held at approximately 20° C. Following this, the arm portion  11  of the dispensing mechanism  10  is moved above the chip disposal unit  40  by the transfer unit  60 , and then disposal of the pipette chip  31  is performed. 
     After primer reagent, enzyme reagent, sample specimen and the dilution sample are discharged into above-mentioned cell unit  54   a , cover closing operation of the cover unit  54   b . of the detection cell  54  is performed. After cover closing operation is completed, temperature of liquid in the detection cell  54  is heated from approximately 20° C. to approximately 65°.  C. to allow amplification of target gene (mRNA) by LAMP (gene amplification) reaction. Then, white turbidity due to magnesium pyrophosphate generated by amplification is detected by turbidimetry. Specifically, detection of turbidity is carried out by detecting (monitoring) turbidity in the detection cell  54  at amplification reaction using LED light source  52   a . and photodiode light receiving unit  52   b . shown in  FIG. 3 . 
     Turbidity data (first measurement results) of the sample specimen and turbidity data (second measurement results) of the dilution sample are transmitted from the gene amplification measuring apparatus  101  to the personal computer  102  in real time. Based on the first measurement results and second measurement results received, the CPU  102   d . of the personal computer  102  judges whether or not gene amplification inhibition is caused. 
     Now, referring to  FIG. 10 , amplification inhibition judgment flow of the CPU  102   d . of the personal computer  102  will be explained. First, in step S 1 , the CPU  102   d . receives from the gene amplification measuring apparatus  101  turbidity data of the sample specimen and the dilution sample, respectively and acquires them. Subsequently, in step  2 , based on the turbidity data of the sample specimen and the dilution sample, the CPU  102   d . displays a graph  113   a . as shown in  FIG. 4  showing relationship between reaction time (min) and turbidity on data browser screen of the display unit  102   c . The graph  113   a . shown in  FIG. 4  is a graph that shows relationship between reaction time (min) of the sample specimen and turbidity. Step S 1  to step S 2 , in which turbidity data are acquired and the graph  113   a  is displayed, are carried out in real time and it is designed so that display of the graph  113   a . is renewed whenever turbidity data is received. When reaction time reaches 16. min, acquisition of data and display of graph  113   a . are completed. 
     Next, in step S 3 , the CPU  102   d . displays rising time “10.7 (min)” of the sample specimen corresponding to turbidity 0.1 in the graph  113   a . in the amplification rising time display column  113   b . In step S 4 , the CPU  102   d . calculates nucleic acid concentrations of the sample specimen and the dilution sample from rising time of each of the sample specimen and the dilution sample and from the analytical curve shown in  FIG. 8 . In step S 5 , the CPU  102   d . displays nucleic acid concentration or range of nucleic acid concentration of the sample specimen (“&lt;2.5E+02” on the screen) on the concentration measurement display column  113   c . Next, in step S 6 , the CPU  102   d . judges presence or absence of amplification inhibition based on nucleic acid concentration of each of the sample specimen and the dilution sample. Specifically, it is judged that amplification inhibition is caused when nucleic acid concentration of a sample specimen is less than 2.5×102. (copies/μl) and nucleic acid concentration of the sample specimen is not less than 2.5×102 (copies/μl). Cases other than this are judged that no amplification inhibition is caused. Then, in step S 7 , the CPU  102   d . displays flag “I” indicating amplification inhibition occurrence in the judgment result display column  113   d . In the meantime, although nucleic acid concentration in the sample is calculated from rising time of the sample corresponding to turbidity 0.1. and the analytical curve shown in  FIG. 8  after reaction time reached 16. min, nucleic acid concentration in the sample may be calculated from rising time of the sample corresponding to turbidity 0.1. and the analytical curve shown in  FIG. 8  at the point when turbidity reaches 0.1. before reaction time reaches 16. min. If this is the case, step S 1  to step S 2 , and step S 3  through step S 7  are subjected to parallel processing. 
     As mentioned above, detection of a target gene (mRNA) is carried out at the reaction detection block  50   a . located at the deepest and at the same time, detection result is displayed on the display unit  102   c . Further, for reaction detection blocks  50   a . at second to fourth from the back, the same target gene detection operations as observed at the reaction detection block  50   a . at the cell setting position  1  are performed sequentially. And, at the reaction detection block  50   a . located at the cell setting position  5  located at fifth from the back, in similar fashion as target gene detection operation at the reaction detection block  50   a . at the cell setting position  1  as mentioned above, the positive control in the container  24  being set in the sample container setting hole  21   a . of the sample specimen setting unit  20  and the negative control in the container  25  being set in the sample container setting hole  21   b  are measured, and judgment is made whether or not detection results at the reaction detection block  50   a . at cell setting positions  1  to  4  are correct. By these operations, one batch processing, in which four sample specimens (including four dilution samples) are processed collectively, is completed. In this way, operations of the gene amplification and analysis system  1  is completed by executing batch processing as many as the predetermined times. 
     According to the present embodiment, as mentioned above, in some cases, it is possible to confirm amplification of a target gene (mRNA), that was once judged to be negative by measurement result by the sample specimen, to be positive from measurement result of a dilution sample, as shown by the graph in  FIG. 6 , by acquiring measurement result (concentration) obtained by a dilution sample of primer and nucleic acid amplification enzyme and sample specimen, in addition to measurement result (concentration) obtained by primer and nucleic acid amplification enzyme and sample specimen. This is attributable to that inhibition substances, which adhered to a target gene and nucleic acid amplification enzyme at the time of measurement by sample specimen and inhibited amplification of the target gene, are liberated in the dilution sample at the time of measurement of the dilution sample. In this case, it is possible to judge that amplification inhibition of the target gene was caused at the time of measurement of the sample specimen from measurement result by the sample specimen with which amplification of the target gene was not confirmed and from measurement result by the dilution sample with which amplification of the target gene was confirmed. As a result, it is possible to acquire an accurate measurement result in which presence or absence of occurrence of amplification inhibition is taken into account, from measurement result by the sample specimen and measurement result by the dilution sample. 
     Further, in the present embodiment, by providing the display unit  102   c . for displaying information relating to amplification inhibition of target gene (mRNA), it is possible for the user to confirm information concerning amplification inhibition (flag “I”) acquired based on measurement result (concentration) by sample specimen and measurement result (concentration) by dilution sample. 
     Further, in the present embodiment, by causing the CPU  102   d . of the personal computer  102  to judge whether or not a target gene (mRNA) is present at the predetermined level or more (positive “+”, negative “−”) based on measurement result by the dilution sample and to output judgment result (flag “I”) of amplification inhibition of presence of the target gene not less than the predetermined level by the CPU  102   d , together with judgment result of amplification inhibition (flag “I”), to the display unit  102   c , it is possible to judge whether or not a target gene is present at the predetermined level or more with measurement result using dilution sample, without being affected by amplification inhibition, even a case where amplification inhibition is caused at the time of measurement of sample specimen, and therefore, it is possible to judge accurately whether or not a target gene is present at the predetermined level or more. Further, users can confirm not only presence or absence of amplification inhibition but also whether or not a target gene is present at the predetermined level or more by outputting judgment result of whether or not a target gene is present at the predetermined level or more, together with judgment result of amplification inhibition to the display unit  102   c    
     Further, in the present embodiment, it is possible to confirm amplification of a target gene (mRNA), that could not be confirmed by measurement result by sample specimen, from measurement result by dilution sample, by using unpurified samples as the sample specimen and the dilution sample, and by acquiring measurement result measured by using the dilution sample in addition to measurement result by the sample specimen, even if a large amount of inhibitory substances are contained in the sample specimen and the dilution sample. As a result, it is possible to acquire accurate measurement result promptly even if unpurified sample specimen and the dilution sample, which do not require time for purifying target gene (mRNA) from sample specimen and the dilution sample, are used. 
     It should be understood that embodiments disclosed herein are exemplifications in all respects and do not constitute a limit. The scope of the present invention is defined by the appended claims, but by not descriptions of the embodiments described above, and all modifications within the appended claims and equivalents are considered to be included within the scope of the present invention. 
     For example, in the above-mentioned embodiment, although a case where the present invention is applied to a gene amplification and analysis system comprising a gene amplification measuring apparatus and a personal computer, the present invention is not limited thereto, and the gene amplification measuring apparatus may be used alone or the gene amplification measuring apparatus may be configured to equip functions of the personal computer. 
     Further, in the above-mentioned embodiment, although such a case is exemplified where flag “I” indicating occurrence of amplification inhibition is displayed when measurement result (concentration) of sample specimen (CK19) is less than 2.5×10 2 . (copies/μl) and at the same time, measurement result (concentration) of dilution sample of the sample specimen (CK19-D) is not less than 2.5×10 2 . (copies/μl), the present invention is not limited thereto, and the flag indicating occurrence of amplification inhibition may be displayed when measurement result of the dilution sample is greater than measurement result of the sample specimen by comparing measurement result (concentration) of the sample specimen to measurement result (concentration) of dilution sample of the sample specimen. 
     Further, in above-mentioned embodiment, although an example where a sample container accommodating the sample specimen and a dilution sample thereof is placed in the sample container setting hole of the sample specimen setting unit, the present invention is not limited thereto, and such an alternative may be made where as the gene amplification measuring apparatus  201  according to the variant of the present embodiment shown in  FIG. 9 , the dilution unit  221  for mounting a container accommodating dilution liquid is provided to the sample specimen setting unit  220 , the dilution liquid is dispensed from the dilution unit  221  to one cell unit  54   b . of the detection cell by the dispensing mechanism  10 , and the dilution sample is produced automatically in the gene amplification measuring apparatus  201 . In this case, it is preferable that, when measurement result of a sample specimen is not less than 2.5×10 2 . (copies/μl), a dilution sample is not produced; and when measurement result of the sample specimen is less than 2.5×10 2 . (copies/μl), the dilution sample is produced. With this consideration, when measurement result of a sample specimen is not less than 2.5×10 2 . (copies/μl), it is apparent that judgment result is positive “(+)” and therefore, it is possible to suppress waste of dilution liquid required for preparation of dilution sample. 
     Further, in the above-mentioned embodiment, although it is judged that nucleic acid amplification product (target nucleic acid concentration) of dilution sample of sample specimen has amplification inhibition of target nucleic acid concentration, when nucleic acid amplification product (target nucleic acid concentration) of sample specimen is abundant, it may be judged that nucleic acid amplification product (target nucleic acid concentration) of dilution sample of sample specimen has amplification inhibition of target nucleic acid concentration when nucleic acid amplification product of dilution sample of sample specimen is more abundant than nucleic acid amplification product proportional to the dilution rate, even when nucleic acid amplification product (target nucleic acid concentration) of sample specimen is insufficient. For example, when nucleic acid amplification product of the sample specimen is 25×10 2 . (copies/μl), nucleic acid amplification product of 10-fold dilution sample, which should be 2.5×10 2  (copies/μl), is 15×10 2 . (copies/μl) and is not proportional to the dilution rate, it may be judged that there is amplification inhibition of target nucleic acid concentration. 
     Further, in the above-mentioned embodiment, although it is judged whether or not the target nucleic acid is present at a predetermined level or more by measuring magnesium pyrophosphate (nucleic acid amplification product) of the sample. In this case, it is preferable to judge whether or not the target nucleic acid is present at a predetermined level or more by measuring target nucleic acid amplification product of the sample. 
     Further, in above-mentioned embodiment, although the sample specimen, the enzyme reagent and the primer reagent of CK19. are dispensed from the sample container  22 , the enzyme reagent container  26  and the primer reagent container  27  to one cell unit  54   a . of the detection cell by the dispensing mechanism  10 , and the dilution sample, the enzyme reagent and the primer reagent of CK19. are dispensed from the sample container  23 , the enzyme reagent container  26  and the primer reagent container  27  to one cell unit  54   b . of the detection cell by the dispensing mechanism  10 . In this case, it is preferable that it is prepared a first sample from the sample specimen, the enzyme reagent and the primer reagent of CK19. and a second sample from the dilution sample, the enzyme reagent and the primer reagent of CK19, and it is dispensed the first sample and the second sample to the cell unit  54   a . and  54   b . of the detection cell by the dispensing mechanism  10 , respectively.