Rapid microorganism testing method and device

Abstract

To provide technologies for rapidly detecting a microorganism in a sample.SOLUTION: Provided is a method for detecting a microorganism in a sample, comprising the steps of: providing a test sample; dividing the test sample to provide at least two partial samples; performing different treatments on a first partial sample and a second partial sample; measuring a marker substance present in the microorganism or secreted by the microorganism in the first partial sample to obtain a first measurement value; measuring the marker substance in the second partial sample to obtain a second measurement value; calculating a threshold value from the first measurement value; and comparing the second measurement value with the threshold value to determine whether or not the microorganism is contained in the sample.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method and device for detecting microorganisms in a sample. [Background technology]

[0002] In sterility tests to check that medicines, cosmetics, food, etc. are not contaminated with bacteria or fungi (hereinafter simply referred to as "bacteria" or "microorganisms"), it is necessary to detect a very small number of bacteria. Conventionally, the culture method used for sterility tests involves culturing bacteria in a medium to increase the number of cells and then detecting them. This method requires culture for more than one day to obtain a sufficient number of bacteria for detection, and culture for more than one week to confirm a negative result, so the time required is a major issue. Therefore, several rapid test methods have been developed in recent years.

[0003] The CO2 detection method is known as a simple testing method, in which a sample is added to a liquid culture medium sealed in a sealed container, and the carbon dioxide produced by the bacteria is detected as the bacteria grow (Patent Document 1).

[0004] Also, a detection method using the adenosine triphosphate (ATP) method is known as a method capable of detecting bacteria with higher sensitivity. The ATP method detects bacterial ATP by bioluminescence caused by a luciferin-luciferase reaction, and can generally detect about 100 CFU (colony forming units) of bacteria.

[0005] For example, Patent Document 2 discloses a method in which a sample is filtered to collect and concentrate bacteria on the filter, and then ATP outside the bacteria is eliminated to remove background ATP, and then ATP inside the bacteria is extracted. The method of Patent Document 2 enables highly sensitive detection of bacteria, and even a few bacteria can be detected. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 6,074,870 [Patent Document 2] U.S. Patent No. 9,290,789 Summary of the Invention [Problem to be solved by the invention]

[0007] When detecting bacteria by quantifying marker substances derived from bacteria, such as metabolites, genes, and substances constituting the bacterial body, if the presence of bacteria can be detected in smaller quantities, rapid testing with high sensitivity will be possible.

[0008] However, since the number of microorganisms to be detected in sterility tests is small (less than 100 CFU), the amount of marker substance is also very small, and in many cases, there is a problem that the marker substance contained in the test sample itself interferes with the detection of the marker substance derived from the bacteria. For example, when the amount of the marker substance contained in the test sample itself is one order of magnitude greater than the amount of the marker substance contained in the bacteria, it is necessary to perform an operation such as enrichment culture of the bacteria until the marker substance contained in the bacteria increases significantly, and then measuring the marker substance.

[0009] In this case, the threshold for bacterial detection is set based on the amount of marker substance contained in the test sample. Ideally, the detection threshold should be set based on the amount of marker substance contained in the test sample itself and its variability. For example, it is common to measure the amount of marker substance in a negative control (sterile test sample) multiple times, calculate the average value a and standard deviation σ, and then calculate the lower detection limit as a + 3.3σ.

[0010] However, the inventors considered that the problem with products using biological raw materials is that the negative control values ​​fluctuate from day to day or from lot to lot. In addition, for small-volume products such as custom-made cell preparations, the amount available for sterility testing is so small that it is not possible to estimate the variability by measuring multiple times.

[0011] Therefore, the present invention provides a technique for appropriately setting a bacteria detection threshold value and quickly detecting bacteria. [Means for solving the problem]

[0012] In order to solve the above problems, the inventors discovered that by dividing a test sample into at least two, treating each divided sample differently so that if bacteria are present, there will be a difference in the amount of marker substance in each sample, measuring the amount of marker substance, using the measured value of one sample to calculate a threshold value, and comparing the measured value of the other sample with the threshold value to determine the presence or absence of bacteria, it is possible to detect bacteria quickly and appropriately.

[0013] Thus, in one aspect, the present invention provides a method for detecting a microorganism in a sample, comprising the steps of: providing a test sample; dividing the test sample to provide at least two aliquots; performing different treatments on the first sub-sample and the second sub-sample; measuring a marker substance present in or secreted by the microorganism in a first partial sample to obtain a first measurement value; measuring the marker substance in a second subsample to obtain a second measurement; calculating a threshold value from the first measurement value; comparing the second measurement value with the threshold value to determine whether the sample contains the microorganism; The present invention provides a method comprising:

[0014] In another aspect, the present invention provides a microbial detection apparatus, comprising: a measurement unit that measures a marker substance present in or secreted by the microorganism in at least two partial samples obtained by dividing the test sample; a memory unit for storing a variation value of the measurement value of the marker substance previously obtained for a sample of the same type as the test sample; Control Analysis Department and Equipped with The control analysis unit, calculating a detection threshold value based on a measurement value of one of the partial samples measured by the measurement unit and the variation value from the memory unit; A measurement value of the separate partial sample measured by the measuring unit is compared with the threshold value to determine whether the microorganism is contained in the test sample. The present invention provides an apparatus for Effect of the Invention

[0015] The disclosed microorganism detection method and device can rapidly detect microorganisms even when there are only a small amount of microorganisms or a small amount of the marker substance to be measured in a sample. Therefore, the present invention is applicable to the fields of pharmaceutical and cosmetic manufacturing, especially cell product manufacturing, food manufacturing, etc. [Brief description of the drawings]

[0016] [Figure 1] 1 is a graph showing an example of measuring the variation in the amount of a cell-derived marker substance (ATP) in a cell sample. [Diagram 2] 1 is an example of a flowchart of a method for detecting bacteria (microorganisms) in a cell sample. [Diagram 3] 1 is a flowchart of a method for detecting bacteria in a cell sample based on the amount of ATP in Example 1. [Figure 4] 1 is a graph showing an example of detection of bacteria in a cell sample. [Diagram 5] FIG. 11 is a schematic diagram of a configuration example of a microbial detection device according to a second embodiment. [Figure 6] 10 is a schematic diagram of a configuration example of a measurement device in a microorganism detection device of Example 2. FIG. [Figure 7] 2 shows an example of a database stored in a storage device of a microorganism detection device. [Figure 8] 13 shows an example of a screen display of an input / output device of the microorganism detection device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention relates to a method and an apparatus for detecting microorganisms. The present invention is characterized by a method for determining a threshold value as a detection standard when detecting microorganisms in a sample based on the amount of a marker substance present in or secreted by the microorganism. The present invention will be described in detail below.

[0018] Figure 1 shows the results of measuring the ATP content of a sterile cultured T cell suspension, which was subjected to the following procedure three times a day, eight times on different days. 6 In order to detect bacteria from a suspension containing bacteria using ATP as a marker substance, ATP derived from cells was removed from the cell suspension as follows. First, a cell lysis solution was added to the cell suspension to lyse the cells, and the cells were filtered through a centrifugal filter. The filter has a pore size of 0.45 μm, and if the sample contains bacteria, the bacteria will be captured on the filter. Next, a cleaning solution was added to the filter and filtered to remove the lysed cells remaining on the filter. Furthermore, an ATP elimination reagent (ATP decomposition enzyme solution) was added to the filter to decompose free ATP for 40 minutes, and the solution was removed by filtration. An ATP extraction reagent (a reagent that destroys bacteria and extracts ATP within the bacteria) was added to this to extract ATP, and the filtrate was obtained in a tube for ATP measurement as a measurement sample after filtering. An ATP luminescence reagent was added to this, and the amount of ATP was quantified from the amount of luminescence. The reagents and filters used in the above operations were sterilized. T cells were a single cell line that was continuously passaged under certain culture conditions.

[0019] Because the samples used here were sterile, the amount of ATP measured (Figure 1) was the ATP derived from the cells that was not completely removed by filtration or the ATP removal reagent. Three aliquots were taken from one cell suspension and measured on the same day, and the standard deviation of the daily variation was 9.4 amol (attomole, 10 -18 The standard deviation of the day-to-day variation was 22 amol, which was larger than the intraday variation. The intraday variation is thought to be caused by variations in sampling, cell removal procedures, and ATP measurement, while the interday variation reflects variations in the state of the cells. Thus, there is a large difference in state from day to day even in samples passaged from the same cell line.

[0020] Therefore, the ideal threshold for bacterial detection is the average ATP measurement value of the sample on the day of testing plus the intraday standard deviation. For example, if 10 times the standard deviation is used to calculate the threshold, the threshold can be set to 63.4 + 10 x 4.7 = 110.4 amol, based on the average measurement value of 63.4 amol and standard deviation of 4.7 amol on the first day. By calculating the threshold in the same way, it is possible to set a threshold value according to the state of the cells on each day, such as 145.1 amol on the second day, 90.1 amol on the third day, etc.

[0021] However, in practice, it is difficult to set such a threshold value. The reason for this is that the presence or absence of bacteria in actual test samples is unknown, and measurements are not purely negative controls. Also, if measurements are taken multiple times for each test sample and the average value and variance are calculated, the number of measurements increases, raising costs. Furthermore, when the amount of target product is small and the amount of test sample available for sterility testing is small, it is difficult to take multiple measurements and calculate the variance in measurements.

[0022] Therefore, in the present invention, the threshold value is set by a method as shown in FIG. First, a standard sample is prepared. The standard sample is of the same type as the test sample. The term "same type" refers to a sample of the same type, for example, a sample containing the same type of cells. For example, in the case of testing a cultured cell sample in the manufacture of biopharmaceuticals, a different lot of the same cell line as the test sample may be used, and multiple lots may be prepared and used. In the case of testing a T cell preparation product made of autologous cells, it is preferable to use T cells from the same subject as the same type, but if the amount of sample available for measurement is limited, an established T cell line, or T cells provided by another subject, may be used. In the case of testing a beverage product such as fruit juice, a product manufactured from raw materials of the same lot, or a different lot, or multiple lots may be used.

[0023] In one embodiment, the standard sample is a sterile sample. In this case, the standard sample is separately subjected to a sterility test using a culture method or the like to confirm its sterility. Sterility testing here refers to the absence of bacteria that grow to a detectable number when culture is attempted under certain sterility test conditions. In reality, there may be live bacteria that do not grow under those test conditions, or live bacteria at concentrations below the lower limit of detection. In this case, if the amount of marker substance possessed by these bacteria is at a level that does not affect the threshold setting by the method described below, or the positive / negative judgment using the threshold, it is considered that the sample can be considered equivalent to sterility. When a sterile or equivalent to sterile sample is used, the measured test target marker substance contains no marker substance derived from bacteria, or only to a level that does not affect the measurement, and the variation of the marker substance derived from the sample can be obtained more accurately.

[0024] In another embodiment, the standard sample is a non-sterile sample. Even in the case of a non-sterile sample, if the mixed bacteria are uniformly dispersed and the marker substance does not increase or decrease during measurement, it can be used as a standard sample. In this case, when the amount of the marker substance in the standard sample is measured, the absolute value is the sum of the value of the standard sample and the value of the bacteria, but the variation is derived from the sample, and it is possible to calculate the standard deviation σ described below. In reality, it is expected that there will be variation in the dispersion of the mixed bacteria and fluctuations in the marker substance, but if the variation resulting from this is sufficiently smaller than the variation of the sample itself (for example, 1 / 10 or less), it can be used as a standard sample. This is useful when the sample to be tested is difficult to sterilize, such as fermented foods or blood samples taken from humans.

[0025] Next, the marker substance to be tested (e.g., ATP) is measured multiple times for the standard sample. It is preferable to measure the standard sample on different days or for multiple lots. From the measurement results, the standard deviation σ is calculated as the value of variation (if measurements are taken on multiple days, the value of intraday variation is calculated excluding interday variation). Next, a coefficient m for calculating the threshold value is set, which will be described later.

[0026] Next, a test sample is prepared and divided into at least two partial samples. These at least two partial samples are subjected to different treatments so that if bacteria are present, there will be a difference in the measured values ​​of the marker substance to be tested between the two. In the example in Figure 2, A is sterilized to reduce the amount of the marker substance derived from bacteria, and B is cultured for a short period of time to increase the amount of the marker substance derived from bacteria. If the difference between the two measured values ​​is greater than the judgment value t calculated from a predetermined variance σ, it is judged as positive for bacteria, and if it is smaller, it is judged as negative for bacteria.

[0027] In this manner, in the present invention, the sample is divided, and each divided sample is subjected to different treatments so that if the target microorganism is present, the amount of the marker substance in the divided samples will differ. The amount of the marker substance is then measured, and the measured value of one sample is used to calculate a threshold value, which is compared with the measured value of the other sample to determine the presence of the microorganism.

[0028] Thus, in one aspect, the present invention provides a method for detecting a microorganism in a sample, comprising the steps of: providing a test sample; dividing the test sample to provide at least two aliquots; performing different treatments on the first sub-sample and the second sub-sample; measuring a marker substance present in or secreted by the microorganism in a first partial sample to obtain a first measurement value; measuring the marker substance in a second subsample to obtain a second measurement; calculating a threshold value from the first measurement value; comparing the second measurement value with the threshold value to determine whether the sample contains the microorganism; The present invention provides a method comprising:

[0029] In the present invention, the "microorganisms" to be detected refer to various kinds of microorganisms including bacteria, actinomycetes, fungi, etc. Specifically, these include microorganisms that are the targets of detection by sterility testing methods in the pharmacopoeias, and pathogenic bacteria and fungi that are the targets of testing in hospital laboratories, etc. For example, Pseudomonas (specifically, Pseudomonas aeruginosa), Propionibacterium (specifically, Proprionobacter acnes), etc. acnes), Staphylococcus (specific examples include Staphylococcus aureus and Staphylococcus epidermidis), Micrococcus, Streptococcus (specific examples include Streptococcus pyogenes and Streptococcus pneumoniae), Enterococcus (specific examples include Enterococcus faecium and Enterococcus faecalis), Neisseria (specific examples include Gonorrhoeae and Neisseria meningitidis), Moraxella, Escherichia (specific examples include Escherichia coli), Shigella (specific examples include Shigella), Salmonella (specific examples include Salmonella typhi, Salmonella paratyphi A, and Salmonella enteritidis), Citrobacter, Klebsiella (specific examples and Examples of bacteria that may be affected include Klebsiella pneumoniae), Enterobacter, Serratia (for example, Serratia marcescens), Proteus, Providencia, Morganella, Yersinia (for example, Yersinia pestis), Vibrio (for example, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio mimicus), Aeromonas, Acinetobacter (for example, Acinetobacter baumannii), Alcaligenes, Agrobacterium, Flavobacterium, and Haemophilus (for example,influenzae), Pasteurella, Francisella, Bordetella (a specific example is Bordetella pertussis), Eikenella, Brucella, Streptobacillus, Actinobacillus, Legionella (a specific example is Legionella pneumophila), Bacillus (examples include Bacillus subtilis, Bacillus anthracis, and Bacillus cereus), Corynebacterium (examples include Corynebacterium diphtheriae), Lactobacillus, Listeria, Erysipelothrix, Nocardia, Actinomyces, Clostridium Clostridium (specific examples include Clostridium perfringens and Clostridium sporogenes), Bacteroides (specific examples include Bacteroides fragilis), Fusobacterium, Mycobacterium (specific examples include Mycobacterium tuberculosis), Campylobacter, Helicobacter (specific examples include Helicobacter pylori), Spirillum, Treponema, Borrelia, Leptospira, Mycoplasma (specific examples include Mycoplasma pneumoniae), Aspergillus (specific examples include Aspergillus niger, Aspergillus braziliensis, The microorganisms to be detected include bacteria and fungi such as Candida brasiliensis and yeast (Candida albicans is a specific example), but other microorganisms can also be detected. In the present invention, even microorganisms that are difficult to detect due to their low ATP content among pharmacopoeia indicator bacteria (e.g., Pseudomonas aeruginosa) can be detected with a short culture period.

[0030] The test sample is not particularly limited as long as it is a sample for which the presence or absence of a microorganism is desired to be determined. The test sample particularly includes biological samples derived from a living organism, samples suspected of being contaminated by a microorganism, and samples for which the presence of a microorganism should be determined to be negative. For example, various samples such as cell samples (e.g., stem cells, T cells, grafts, cell culture fluid, cell culture supernatant, etc.), body fluid samples (e.g., whole blood, serum, plasma, urine, bone marrow fluid, semen, breast milk, amniotic fluid, saliva, etc.), pharmaceutical or cosmetic samples (e.g., injections, drips, eye drops, raw materials, manufacturing intermediates, container cleaning fluids, etc.), environmental samples (e.g., clean room air, surfaces, etc.), culture medium samples, food samples (e.g., foods, beverages, feed, supplements, intermediates in the manufacturing process, etc.), water samples (e.g., seawater, river water, industrial water, etc.), and soil samples can be used. In addition, in the case of samples derived from a living organism, the origin of the test sample is not particularly limited, and it can be derived from any biological species. For example, the test sample may be a sample derived from at least one of various types of living organisms, such as animals, plants, and insects.

[0031] In the present invention, "detection" means determining whether or not a microorganism is present in a test sample, estimating the amount of a microorganism in a test sample, determining that a microorganism is not present in a test sample (negative for a microorganism), etc. The concentration of a microorganism that may be contained in a test sample is not particularly limited.

[0032] Moreover, the "marker substance" to be measured means a substance present in or secreted by the microorganism to be detected, and is a substance whose amount or concentration in a sample can be measured. The marker substance varies depending on the type of microorganism to be detected, the type of sample, the measuring device, etc., and can be appropriately selected by a person skilled in the art. For example, ATP, endotoxin, nicotinamide adenine dinucleotide (reduced NAD(P)H, etc.), genes (DNA, RNA, miRNA, etc.), proteins (β-galactosidase, NADH-quinone oxidoreductase, other enzymes, surface proteins expressed on the surface of microorganisms, etc.), sugar chains, membrane lipids, metabolites, etc. can be mentioned. Methods and means for measuring such marker substances are known in the art.

[0033] In this method, a test sample is first prepared. When the test sample is a liquid sample, it can be used as is, or after dilution or concentration with a solvent. When the test sample is a solid sample, it can be suspended in a solvent, homogenized with a grinder, or the like, or the supernatant obtained by stirring with a solvent can be used. Examples of the solvent include distilled water, physiological saline, phosphate buffer, Tris buffer, etc. Furthermore, it is also possible to use a filter in which a sample is filtered and microorganisms are captured. Such a filter is not particularly limited as long as it has a size that can capture microorganisms.

[0034] Next, the test sample is divided to prepare at least two partial samples. For example, the test sample is divided into two. The divided partial samples are placed in separate containers. In this specification, a case where the sample is divided into two to prepare a first partial sample and a second partial sample is described in detail, but if the amount of the test sample is large, a third partial sample, a fourth partial sample, etc. may be prepared and can be processed in the same manner.

[0035] Next, different treatments are performed on the divided partial samples. That is, different treatments are performed on the first partial sample and the second partial sample. Here, different treatments refer to treatments that cause a difference in the amount or concentration of the marker substance to be measured after the presence of microorganisms in the test sample, and the treatment operation may be different, the treatment conditions (time, temperature, added reagents, etc.) may be different, the number of treatment operations may be different, or one may be treated and the other may be untreated. In one embodiment, a treatment that inhibits the measurement of the marker substance is performed on the partial sample (for example, the first partial sample). Such treatments include, but are not limited to, sterilization treatments (for example, heat treatment, ultraviolet ray / gamma ray / electron beam treatment, treatment with a bactericide, etc.), bacteriostatic treatments (for example, heat treatment, treatment with a bacteriostatic agent, etc., treatments that suppress the growth of live bacteria), and when the marker substance is a protein, proteolysis treatments or treatments with antibodies that inhibit detection. In one embodiment, a treatment is performed on the partial sample (for example, the second partial sample) to increase the amount of the marker substance. The treatment that increases the amount of the marker substance is a treatment that is preferably performed when the amount of the marker substance present in the microorganism is small or when there is a large variation due to daily fluctuations. Such treatment includes, but is not limited to, culture under enrichment conditions. The medium used for culture varies depending on the type of microorganism to be detected and the type of test sample, but for example, media used in existing sterility testing methods (e.g., soybean casein digest (SCD) medium, Sabouraud glucose liquid medium, liquid thioglycolic acid medium, lactose bouillon, etc.) can be used. Those skilled in the art can select the appropriate medium depending on the type of test sample, the type of microorganism that may be present, and the expected amount. The culture can be performed under conditions commonly used in the technical field (temperature, time, aerobic or anaerobic, static or shaking). Specific culture conditions vary depending on the type of target microorganism, the type of test sample, the type of medium used, etc., and can be appropriately set by those skilled in the art. The culture time is specifically 30 minutes to 48 hours, preferably 1 to 30 hours, and more preferably 1 to 24 hours.The incubation time varies depending on the amount and type of microorganisms to be detected, the type of test sample, the culture medium used, the incubation temperature, and the like.

[0036] It is preferable to set the different treatments so that the amount of the marker substance in the first partial sample is less than the amount of the marker substance in the second partial sample. In a preferred embodiment, the first partial sample is subjected to a sterilizing or bacteriostatic treatment, and the second partial sample is cultured under enrichment conditions. In another preferred embodiment, the first partial sample is subjected to a sterilizing or bacteriostatic treatment, and the second partial treatment is not treated (untreated). In yet another preferred embodiment, the culture time of the first partial sample is set shorter than the culture time of the second partial sample.

[0037] In one embodiment, the treatment of the second partial sample includes a treatment for increasing the amount of the marker substance, and the method further includes a step of determining the time of the treatment for increasing the amount of the marker substance of the microorganism based on the known amount of the marker for the microorganism, the known growth rate of the microorganism, and the variation σ described below. Since the doubling time and the amount of the marker substance of the microorganism differ depending on the type of the microorganism, it is desirable to determine the time of the treatment depending on the type of the microorganism.

[0038] Next, the marker substance is measured in the first partial sample to obtain a first measurement value. Furthermore, the marker substance is measured in the second partial sample to obtain a second measurement value. These steps may be performed in parallel or successively. Performing some or all of the steps in parallel allows the test to be completed in a shorter time. The process for changing the amount of the marker substance in each partial sample may be performed in parallel, and the measurement of the marker substance may be performed successively. Either the first measurement value or the second measurement value may be obtained first. Furthermore, the second measurement value may be obtained after obtaining the first measurement value and calculating the threshold value described below.

[0039] The method of measuring the marker substance varies depending on the type of the marker substance, and a person skilled in the art can measure the marker substance appropriately. For example, when the marker substance is ATP, the measurement of ATP can be performed by a known ATP luminescence measurement method. Specifically, using luciferase and luciferin, which chemically react with ATP to generate light, the luminescence generated by the reaction of ATP in the cells with luciferase and luciferin is measured, and the ATP value is determined from the amount of luminescence. Such ATP measurement can be easily performed by a person skilled in the art using a commercially available ATP detection reagent or kit. As a pretreatment, it is preferable to remove ATP other than ATP derived from microorganisms, for example, by adding ATP decomposition enzyme to the sample. Next, the ATP in the cells is extracted using a known ATP extraction solution (e.g., trichloroacetic acid, surfactant, lysozyme, etc.). Then, the extracted ATP is made to emit light using a luciferin-luciferase luminescence reaction. Specifically, a luminescence reagent containing luciferin and luciferase is added to the sample. The luminescence reagent reacts with the extracted ATP to generate luminescence, and the amount of luminescence is measured. Luminescence reagents are also known in the art and are not particularly limited. Luminescence measurement can be performed by luminescence measurement methods and light detectors known in the art, for example, using a luminometer, a spectrophotometer, or a luminescence plate reader. Based on the measurement of the amount of luminescence, the ATP value in the sample is obtained.

[0040] When the marker substance is endotoxin, the measurement of such substances is well known in the art, and many detection kits and detection systems are commercially available. Preferably, a method or kit capable of measuring endotoxin by optical measurement is used.

[0041] Even when the marker substance is a gene (DNA, RNA, miRNA, etc.), the measurement of gene amount is well known in the art, and many techniques are known that apply the principles of PCR (polymerase chain reaction), hybridization, etc. Those skilled in the art can measure the gene as a marker substance by an appropriate method depending on the gene to be measured. Kits containing primers that PCR amplify the common region of 16S ribosomal DNA of bacteria or fungi are commercially available, making it possible to detect bacteria by PCR. After detection of bacteria, it is also possible to identify the species of bacteria by sequencing the PCR product.

[0042] When the marker substance is a protein (such as a surface protein, β-galactosidase or other enzymes), the marker substance can be measured using an antigen-antibody reaction, an enzyme reaction, or the like, depending on the type of protein. Such protein measurement is also well known in the art, and many detection kits and detection systems are commercially available. Preferably, a method or kit capable of measuring proteins by optical measurement is used.

[0043] When the marker substance is a metabolite, the measurement can be performed by a means for measuring a physical or chemical property specific to the metabolite, for example, a means for measuring an accurate molecular weight or an NMR spectrum. Analytical devices such as a mass spectrometer (MS), an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, and a liquid chromatography mass spectrometer (LC / MS) are known as means for measuring metabolites. When the metabolite is ATP, it can be measured by luminescence measurement using the above-mentioned ATP measurement kit. In addition, when the metabolite is a reactive chemical substance, such as nicotinamide adenine dinucleotide, for example, reduced nicotinamide adenine dinucleotide (NAD(P)H), there are a method of measuring by fluorometric measurement by adding a dye that reacts with the substance and changes the fluorescence wavelength, and a method of measuring the active oxygen by chemiluminescence by adding a reagent that reacts with the substance and generates active oxygen.

[0044] Next, a threshold value is calculated from the first measurement value. The threshold value is important in determining the presence of microorganisms based on the difference in the amount of the marker substance in the partial samples subjected to the above-mentioned different treatments, and is calculated taking into consideration the variation caused by the variation in the amount of the marker substance in the test sample and the measurement variation of the amount of the marker substance. The variation is preferably determined based on the variation within the measurement day. This variation can be determined in advance using a sample of the same type as the test sample or a part of the test sample, or can be determined in parallel with the present method. Note that, if the variation caused by the variation in the amount of the marker substance in the test sample is sufficiently smaller than the measurement variation of the amount of the marker substance, the threshold value may be calculated using only the measurement variation of the amount of the marker substance.

[0045] For example, a case will be described in which a sample of the same type as the test sample is used to determine the variability of the sample, and then the threshold value is calculated. preparing a standard sample of the same type as the test sample; measuring the marker substance in the standard sample multiple times; Calculating the variation σ of the obtained plurality of measurement values; calculating an average value a of the first measurement values; Calculating the threshold value t as t=a+m·σ, where m is an arbitrary coefficient. Further includes:

[0046] The standard sample is as described above, and may be, for example, a different lot of the same product as the test sample, and multiple lots may be prepared and used. The standard sample may be one that has been confirmed to be sterile, or it may be a non-sterile sample.

[0047] Next, the marker substance is measured multiple times in the standard sample. The multiple measurements are preferably performed on the same day to grasp the intraday variation, but the variation can be understood in more detail by performing measurements on different days or for multiple lots. The variation σ (standard deviation) is calculated from the multiple measured values ​​obtained.

[0048] The average value a of the first measurement values ​​described above is calculated, and the threshold value is calculated as t=a+m·σ. Here, the coefficient m represents how many times the variation σ the difference between the measurement value of one partial sample (e.g., the first measurement value) and the measurement value of another partial sample (e.g., the second measurement value) must be to determine that a microorganism is present in the sample. For example, if the difference is 10 times or more the variation σ to determine that a microorganism is present, the coefficient m is 10. m is an arbitrary value, but is 3 or more, which is the detection limit, preferably 6 or more, and more preferably 9 or more. The larger the value of m, the lower the false positive rate, but on the other hand, the lower the sensitivity (the rate at which positive samples are determined to be positive), so it is desirable for the value to be 20 or less.

[0049] Although a specific example for calculating the threshold value has been described, the method is not limited to the above-described method and may also calculate the threshold value using other methods.

[0050] Thereafter, the second measurement value is compared with the above-mentioned threshold value to determine whether or not the sample contains microorganisms. That is, if the second measurement value is smaller than the above-mentioned threshold value, it is determined that no microorganisms are present in the sample (negative for microorganisms), and if the second measurement value is equal to or greater than the above-mentioned threshold value, it is determined that microorganisms are present in the sample (positive for microorganisms). Alternatively, for example, if the coefficient m is 5 to 10 and the second measurement value is the same or nearly the same as the above-mentioned threshold value, remeasurement may be performed. If measurements are performed multiple times on the second partial sample, the average value of the second measurement values ​​is used for the determination.

[0051] The method makes it possible to rapidly and / or sensitively detect microorganisms even when only small amounts of microorganisms or marker substances to be measured are present in a sample.

[0052] Furthermore, the present invention provides a microbial detection device for carrying out the above-mentioned method. Specifically, the microbial detection device according to the present invention comprises: a measurement unit that measures a marker substance present in or secreted by the microorganism in at least two partial samples obtained by dividing the test sample; a memory unit for storing a variation value of the measurement value of the marker substance previously obtained for a sample of the same type as the test sample; Control Analysis Department and Equipped with The control analysis unit calculating a detection threshold value based on a measurement value of one of the partial samples measured by the measurement unit and the variation value from the memory unit; A measurement value of the separate partial sample measured by the measuring unit is compared with the threshold value to determine whether the microorganism is contained in the test sample. It is something.

[0053] The measurement unit includes a means for measuring at least the marker substance. Such a measurement means varies depending on the type of the marker substance to be measured, and examples thereof include a photodetector (luminometer, spectrophotometer, luminescence plate reader, etc.) and a mass spectrometer. The measurement unit is configured to measure the marker substance for each of the multiple partial samples. For example, the measurement unit may include multiple measurement means, or multiple partial samples may be measured by one measurement means.

[0054] The storage unit stores at least the value of the variation of the measurement value of the marker substance, which is previously obtained for a sample of the same type as the test sample. The storage unit may store, for each type of marker substance, the measurement value (actual measurement value) of the marker substance when calculating the stored variation value, the measurement method and measurement conditions of the marker substance, the type of test sample, etc. In addition, the measurement value of the marker substance in the test sample newly measured by this device, the measurement method and measurement conditions, the type of test sample, the judgment result, etc. may be stored as needed.

[0055] The control and analysis unit is not particularly limited as long as it can control the entire device or the operations of each component (measurement unit, storage unit, etc.) and / or execute analysis, and is generally a computer. The control and analysis unit may be arranged or integrated in connection with other components (measurement unit, storage unit, etc.), or may be connected via a wired or wireless network.

[0056] The control and analysis unit calculates a detection threshold based on a measurement value of one of the partial samples measured by the measurement unit (e.g., a first measurement value of a first partial sample) and a variation value from the memory unit, and compares a measurement value of another partial sample measured by the measurement unit (e.g., a second measurement value of a second partial sample) with the threshold to determine whether or not the test sample contains a microorganism. The specific determination method is the same as the above-mentioned microorganism detection method.

[0057] The apparatus may further include a sample processing section having at least two sample containers, in which different processes are performed on at least two partial samples. For example, in addition to the sample containers, the sample processing section may include a means for separating cells and microorganisms contained in the sample (such as a filter or a centrifuge), a container for collecting a marker substance to be measured, a temperature control device, and the like. The sample processing section may also include a means for dividing the test sample (such as a dispensing pipette).

[0058] The device may further include an input unit and / or an output unit. The input unit is used to input at least one piece of information selected from the group consisting of the type of test sample, the type of marker substance, and the measurement method of the marker substance to the control and analysis unit. The output unit outputs, for example, the result of the determination made by the control and analysis unit.

[0059] The present device may also include a portion for holding a reagent used in the measurement section and a means for introducing the reagent. For example, when measuring ATP as a marker substance, the device may include a portion for holding an ATP detection reagent (ATPase, ATP extract, ATP luminescence reagent, etc.) and a means for introducing the reagent into each partial sample.

[0060] The device may include a mechanism for transporting a sample or a container containing a sample or a container containing a marker substance between the sample processing section and the measurement section.

[0061] This device can rapidly detect microorganisms in a sample, and has the advantage that it can rapidly detect microorganisms even when the sample contains only a small amount of microorganisms or when the amount of the marker substance to be measured is small. EXAMPLES

[0062] The present invention will be described in more detail below with reference to examples and drawings, although the present invention is not limited to the following examples.

[0063] [Example 1] As a specific example, detection of bacteria in a T cell culture medium using the method of the present invention is shown in Figure 3. In this example, ATP was measured as the marker substance to be tested.

[0064] First, as a standard sample (T cell sample), 10 T cells were 6 The T cell culture fluid containing the cells was measured three times a day, and the amount of ATP was measured on several different days, and the standard deviation within a day was calculated to be σ = 9.4 amol (Figure 1). The method was the same as that explained in Figure 1, and included cell lysis treatment, capture of bacteria on a filter, activity elimination reaction (ATP elimination reaction), extraction of ATP from the bacteria, ATP measurement, and calculation of the standard deviation (variation) σ (flow on the left side of Figure 3).

[0065] Next, as a test sample (subject T cell sample), 34 CFU of P. aeruginosa and 2 × 10 T cells were 6A T cell culture solution containing 1000 cells was prepared. This was then subjected to cell lysis treatment in the same manner as the standard sample, and divided equally into two portions, A and B, which were then added to separate centrifugal filters and filtered. A washing solution was then added to each filter, and the remaining lysed cells were removed.

[0066] Next, filter A was sterilized by heating. Next, an ATP elimination reagent (ATPase solution) was added to both filters and incubated at 35°C to decompose free ATP over 180 minutes, while simultaneously culturing live bacteria present in filter B.

[0067] Next, the liquid was removed by filtration, and an ATP extraction reagent (a reagent that destroys bacteria and extracts intracellular ATP) was added to this to make a bacterial ATP solution, which was then filtered to obtain a filtrate in a tube for ATP measurement. An ATP luminescence reagent was added to this, and the amount of ATP was quantified from the amount of luminescence.

[0068] By subjecting A to sterilization treatment, the bacteria in the partial sample of A are destroyed, so the measured value a of A does not include ATP derived from the bacteria, but only ATP derived from the cells is measured. In addition, the bacteria in the partial sample of B grow in the ATP elimination reagent, increasing the amount of ATP, so the measured value b of B includes ATP derived from the bacteria and ATP derived from the cells.

[0069] The measurement results are shown in the graph in Figure 4. 34 CFU of bacteria and 2 x 10 T cells 6 In the case of a sample containing bacteria, the measured value a of A was 22 amol, and the measured value b of B was 123 amol (right side of Figure 4). Here, the value a is used instead of the average ATP amount of the test sample itself, which is necessary when calculating the detection threshold, and the standard deviation σ measured in advance using a standard sample is used as the value of variation. If it is set that a difference between the values ​​of A and B is 10 times or more than σ, the result is a positive result for bacteria (i.e., the coefficient m is set to 10), the threshold value t is calculated as a + m × σ = 22 + 10 × 9.4 = 116 amol. Since the value b of B is greater than the threshold value t, it is determined to be positive for bacteria. Here, for example, if the threshold value t = b, it may be possible to retest as a suspected positive result.

[0070] A similar test was performed using 2 × 10 T cells without bacteria. 6 When the test sample was a T cell culture fluid containing bacteria, the value of a was 17 amol and the value of b was 33 amol (left side of Figure 4). Similarly, the threshold was calculated to be 111 amol, and since the value of b was smaller than this value, it was determined to be negative for bacteria.

[0071] Here, in the calculation method of the threshold value t=a+m·σ, the coefficient m may be set to 3.29 or 3 as a calculation method of the detection limit. This is the value when the number of measurements when calculating σ is sufficient, and if n=3 measurements are made, m should be about 6. In addition, since the threshold value is calculated using the value a of a single measurement of partial sample A instead of the average value of multiple measurements, it is preferable to have a likelihood of about 3σ for correction. Therefore, it is desirable to set the m value to 6 or more, more preferably 9 or more. The larger the value of m, the lower the false positive rate, but on the other hand, the lower the sensitivity (the rate of positive samples that are determined to be positive), so it is desirable to set it to 20 or less.

[0072] The amount of the marker substance possessed by the bacteria in the partial sample of B must be at least m·σ higher than that of A. When the amount of the marker substance in the bacteria is low or when σ is large due to daily variation, the false positive rate can be reduced by increasing the value of b through enrichment culture.

[0073] In this example, partial sample A was sterilized and enrichment culture was performed on B, but if it is known that there is a difference in the amount of bacterial-derived ATP between A and B that is sufficiently larger than σ even without enrichment culture, such as when the target lower limit of bacterial detection is high, enrichment culture may not be performed on B. In this case, the activity elimination reaction can be shortened to, for example, 30 minutes for A and B, allowing the presence or absence of bacteria to be determined more quickly.

[0074] Moreover, the same purpose can be achieved by carrying out a process for blocking detection of the detection target substance instead of sterilizing the partial sample A. For example, when an antigen on the surface of a bacteria is detected by an antigen-antibody reaction, a process for decomposing the antigen or a process for adding an antibody that binds to the antigen and inhibits detection by a detection reagent can be adopted.

[0075] Alternatively, a method can be adopted in which partial sample A is not subjected to sterilization treatment or the like, and the ATP elimination reaction of A is carried out for 30 minutes, while the ATP elimination reaction of B is carried out for 180 minutes, thereby creating differences in the bacterial multiplication times.

[0076] It is preferable to process partial samples A and B in parallel. This is because, under suitable conditions, the live bacteria captured on the filter will grow or produce ATP on the filter, and the amount of ATP will change over time. Also, processing A and B in parallel reduces processing time and enables more rapid testing, rather than processing A and B sequentially. Also, the time difference between the start of processing A and B is preferably within 10 minutes. This is because bacteria such as Escherichia coli, which divides quickly, require about 10 to 20 minutes to divide, and accurate measurement becomes difficult if the time difference is large.

[0077] When partial samples A and B are not processed in parallel, but are processed partially or entirely in series, it may be necessary to take measures to prevent unexpected increases in live bacteria during processing from affecting the judgment results. For example, by starting processing B after sterilization processing in A, it is possible to prevent bacteria from multiplying in A and increasing the measured value. Conversely, if the sample contains a substance that inhibits bacterial growth, such as an antibiotic, filtering and washing processing of B first can be performed to remove the antibiotic, allowing bacteria to multiply in B.

[0078] The amount of ATP contained in bacteria differs depending on the species. For example, it is 1 to 10 amol per CFU for Staphylococcus (bacteria belonging to the genus Staphylococcus), and 50 to 500 amol for Candida (fungi belonging to the genus Candida). The doubling time of bacteria (the time it takes for the number of bacteria or the marker substance to be measured to double) also differs depending on the species, being 10 to 30 minutes for Staphylococcus and 1 to 2 hours for Candida. Based on this information, the enrichment culture time can be set.

[0079] First, the amount of ATP contained and the cell division time of the bacteria to be detected are measured and stored in a database. Next, the value of m·σ used to calculate the threshold value is calculated. In the example of T cells in this embodiment, it is 10×9.8=98 amol. When the target detection limit is 30 CFU, 15 CFU are present in the partial sample B and grow during the enrichment culture. For example, when detecting Staphylococcus aureus, the amount of ATP derived from bacteria in the partial sample B before the enrichment culture is 1 amol / CFU×15 CFU=15 amol, which is lower than the threshold value. If this increases with a doubling time of 30 minutes, it will reach 120 amol after 1.5 hours of culture. Therefore, it is found that an enrichment culture time of 1.5 hours is required. In practice, it is desirable to set the enrichment culture time to about 2 to 3 hours with a margin, taking into account the variation when dividing the test sample of 30 CFU, the enrichment culture conditions, and the variation between strains.

[0080] In this way, by setting in advance the amount of the marker substance to be measured, which is derived from bacteria, and the rate of bacterial growth, it is possible to set the time for enrichment culture.

[0081] [Example 2] An example of the configuration of a microbial detection device according to the present invention is shown in Fig. 5. A measuring device 1, an input / output device 2, and a storage device 3 are connected to a computer 4. An example of the configuration of the measuring device (sample processing unit 13 and measuring unit 16) is shown in Fig. 6. A case in which ATP in a sample is measured to determine the presence or absence of bacteria will be described.

[0082] The user uses the input / output device 2 to select the type of sample, the coefficient m used to calculate the threshold value, and the measurement method. Furthermore, the user places a cleaning reagent, an ATP elimination reagent, an ATP extraction reagent, and a luminescence reagent in the reagent holder 5 in the measuring device.

[0083] The user also places the waste liquid container 6 and the sample container 7 with the filtration filter 9 in separate temperature control units 8 (8A, 8B) for the two sets of partial samples A and B. The temperature control units 8A and 8B control the temperature of the filter 9 and its surroundings, and the temperatures are set independently. The filter 9 has a pore size of 0.45 μm or less, is capable of capturing bacteria, and is sterile.

[0084] For example, the user divides the test sample into two equal portions and adds them to each sample container 7. The sample container 7 and waste liquid container 6 are set in a centrifuge rotor 10, and filtration is performed by centrifugation, with the bacteria captured and concentrated on the filter, and the solvent is collected in the waste liquid container 6. By removing the solvent, substances that inhibit the action of reagents to be added later and substances that inhibit the growth of bacteria are also removed, enabling highly sensitive measurements.

[0085] Here, the sample container 7 and the waste liquid container 6 are placed on the centrifuge rotor 10, but a separate tube holder may be provided to transport the sample container 7 and the waste liquid container 6 to the centrifuge rotor when centrifugal filtration is performed. Also, instead of centrifugal filtration, filtration can be performed by other means, such as suction filtration or pressure filtration.

[0086] Next, a cleaning reagent for cleaning the filter is added using a dispensing device 11, and then removed by centrifugal filtration. This cleaning reagent is selected from solutions that do not damage bacteria and can clean the sample matrix, such as a buffer solution, a bacterial enrichment medium, a surfactant solution, and an ATP elimination reagent.

[0087] Next, the filter is heated by the temperature control unit 8A to kill the bacteria on the filter. The temperature is about 60 to 80° C., and the treatment is carried out for about 1 to 10 minutes.

[0088] Next, the ATP elimination reagent is added to both sample containers 7 of A and B using the dispensing device 11, and the extracellular free ATP remaining on the filter is eliminated. At this time, under appropriate conditions, the live bacteria on the filter 9 of B grow, and the ATP derived from the bacteria increases. When the elimination of extracellular ATP and the proliferation are performed simultaneously in this way, a substance or culture medium that serves as a nutrient source to promote the proliferation of bacteria may be added to the ATP elimination reagent. Both temperature control units 8A and 8B are set to a temperature of about 35°C, which is suitable for the ATP elimination reaction and the proliferation, and the reaction is performed for a certain period of time. The conditions of this reaction (temperature and time) can be determined in advance as the proliferation time required to detect the standard strain by testing it with the target number of bacteria. After the reaction, the ATP elimination reagent is removed by centrifugal filtration.

[0089] Next, the sample container 7 is moved to a measurement sample recovery container 12. An ATP extraction reagent is added to the sample container 7 to extract the intracellular ATP. After that, the extract is centrifuged by the centrifugal rotor 10 to recover the extract in the recovery container. After collection, the measurement sample collection container 12 is transported from the sample processing section 13 to the luminescence measurement section 16, and the sample container 7 is removed. A luminescence reagent is added to the extract by the dispensing device 14, and the generated luminescence is measured by the photodetector 15.

[0090] Here, the sample collection container 12 is transported from the centrifuge rotor 10 to the photodetector 15, but the photodetector 15 may be positioned below the centrifuge rotor 10 to measure the luminescence on the spot. In that case, the sample processing section and the measurement section are integrated.

[0091] The computer 4 retrieves the measurement variability value σ corresponding to the type of sample selected by the user and the marker substance to be measured (ATP in this embodiment) from the database in the storage device 3, and calculates the threshold value t=a+m·σ from the m value set by the user and the measurement value a of sample A measured by the photodetector 15. The measurement value b of sample B is compared with the threshold value t, and if b is larger, the result is determined to be positive for bacteria, and if b is smaller, the result is determined to be negative for bacteria. The storage device stores the sample name, the measurement values ​​(a, b), σ, m, t, and the determination result, and outputs the determination result to the input / output device (monitor) 2. If necessary, the false positive rate and / or the false negative rate may be output.

[0092] With the above-described device configuration, an optimal detection threshold can be set for each test sample, enabling more rapid detection of bacteria.

[0093] [Example 3] FIG. 7 shows an example of a database used in the device according to the present invention. The database records in association with the type of sample, the substance to be measured (marker substance), the measurement method, the maximum measurement value, the variance σ of the measurement value, and the data used to calculate these values. The measurement method is the method for sample pretreatment and measurement, and includes information such as the type of reagent, the amount added, temperature conditions, reaction time, filtration conditions, and photodetector settings.

[0094] The measurement substance is not limited to one type, and may include a plurality of types of substances. The measurement substance can be appropriately selected according to the type of bacteria to be detected and the type of test sample. For example, substances that can be quantified by luminescence measurement include ATP, endotoxin, β-galactosidase, etc. If a reagent that can quantify the amount and / or activity of these substances by luminescence measurement is used, it is possible to detect bacteria using various substances with one device. It is also possible to quantify bacteria-derived substances using fluorescence measurement, or to quantify bacteria-derived genes by quantitative PCR.

[0095] When measuring a test sample, the user specifies the sample and the substance to be measured. The computer calculates the threshold value using the corresponding σ value from the database. By saving the maximum value of a in the database, it is possible to display a warning on the output screen (or issue an audio warning) indicating a suspected sample processing error when the measured value a of sample A exceeds this maximum value.

[0096] [Example 4] FIG. 8 shows an example of an input / output screen used in the device according to the present invention. On the Sample analysis screen, you can select the sample name, sample type, and measurement method, and then instruct the system to perform the measurement. After the measurement, the automatically calculated detection threshold, the measurement value of sample B (b), and the judgment result are displayed.

[0097] On the measurement method screen, you can set the m value required to calculate the threshold. Also, the σ value to be used is read from the database. You can also set the temperature for the pretreatment and culture.

[0098] The measurement result screen displays the measurement method, sample type, σ value, m value, measurement results (a and b), calculated threshold value, measurement execution date and time (Start and End), judgment result, false positive rate, etc. This information is stored in the storage device. [Explanation of symbols]

[0099] 1. Measurement equipment 2 Input / Output Devices 3 Storage device 4. Computer 5 Reagent holder 6 Waste container 7. Sample Container 8 Temperature control section (8A, 8B) 9 Filters 10 Centrifuge rotor 11 Dispensing device 12 Measurement sample collection container 13 Sample processing section 14 Dispensing device 15 Photodetector 16 Measurement section

Claims

1. 1. A method for detecting a microorganism in a sample, comprising: providing a test sample; dividing the test sample to provide at least two aliquots; performing different processes on the first sub-sample and the second sub-sample; measuring a marker substance present in or secreted by the microorganism in a first partial sample to obtain a first measurement value; measuring the marker substance in a second aliquot to obtain a second measurement; calculating a threshold value from the first measurement value; comparing the second measurement with the threshold value to determine whether the sample contains the microorganism; Including, preparing a standard sample of the same type as the test sample; measuring the marker substance in the standard sample multiple times; Calculating the variation σ of the obtained plurality of measurement values; calculating an average value a of the first measurement values; The method further comprising the step of calculating the threshold value t as t=a+m·σ, where m is an arbitrary coefficient.

2. The method of claim 1 , wherein the homogenous sample is a sterile sample.

3. The method of claim 1 , wherein the treatment of the first subsample comprises a treatment that inhibits measurement of the marker substance.

4. The method of claim 1 , wherein the treatment of the second aliquot comprises a treatment to increase the amount of the marker substance.

5. The method of claim 1, further comprising determining a treatment time for increasing the amount of the marker substance of the microorganism based on a known marker amount for the microorganism, a known growth rate of the microorganism, and a variability σ.

6. 2. The method of claim 1, wherein the sample is selected from the group consisting of a cell sample, a pharmaceutical or cosmetic sample, a culture medium sample, a body fluid sample, an environmental sample, a food sample and a water sample.

7. The method of claim 1 , wherein the marker substance comprises at least one selected from the group consisting of ATP, endotoxin, nicotinamide adenine dinucleotide, genes, and β-galactosidase.

8. A microbial detection device, comprising: a measurement unit that measures a marker substance present in or secreted by the microorganism in at least two partial samples obtained by dividing the test sample; a memory unit for storing a variation value of the measurement value of the marker substance previously obtained for a sample of the same type as the test sample; Control Analysis Department and Equipped with The control analysis unit calculating a detection threshold value based on a measurement value of one of the partial samples measured by the measurement unit and the variation value from the memory unit; A measurement value of the separate partial sample measured by the measuring unit is compared with the threshold value to determine whether the microorganism is contained in the test sample. It is a device.

9. The apparatus of claim 8 , wherein the measurement portion comprises a photodetector.

10. Sample Processing Unit Having at Least Two Sample Containers - Patent application Further equipped with The device according to claim 8 , wherein the at least two aliquot samples are subjected to different processing in sample containers of the sample processing section.

11. an input unit for inputting at least one piece of information selected from the group consisting of the type of the test sample, the type of the marker substance, and a measurement method for the marker substance into the control and analysis unit; The apparatus of claim 8 further comprising:

12. an output section for outputting the result of the determination in the control analysis section; The apparatus of claim 8 further comprising:

Images