Method for using digital PCR to accurately measure bacterial counts

Abstract

To utilize digital PCR to accurately measure bacterial counts.SOLUTION: Digital PCR-based, accurate measurements of bacterial counts can be achieved by setting rpoB gene as a target gene and utilizing: a primer pair that comprises a rpoB forward primer or a primer mixture containing the rpoB forward primer, and a rpoB reverse primer or a primer mixture containing the rpoB reverse primer; and a rpoB probe mixture.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for quantitatively testing bacteria using a digital PCR method. [Background technology]

[0002] Currently, biomarkers that reflect the severity of infection include procalcitonin, presepsin, body temperature, white blood cell count, and CRP. Although these biomarkers correlate to the severity of infection to some extent, they do not necessarily accurately reflect the severity at that time.

[0003] Therefore, if it were possible to accurately measure the "number of pathogenic bacteria (number of bacteria / mL) in a patient sample," the number of bacteria could be used as a new biomarker for accurately determining the severity of an infectious disease. The present inventors have proposed a method for quantifying bacteria using real-time PCR with specific primers (Patent Document 1), and a method for quantifying bacteria accurately and with high sensitivity using PCR with a specific DNA polymerase (Patent Document 2).

[0004] Furthermore, conventional microbial testing methods prescribed in the Pharmacopoeia require more than a week (7-14 days) to determine sterility. For example, as shown in Figure 1, the conventional method (the most standard method) involves mixing a sample into a liquid culture medium and visually checking whether the medium becomes turbid. However, because a large number of bacteria is required to cause the medium to become turbid, a 14-day incubation period is required to confirm sterility. The BacteAlert method, a rapid sterility testing method prescribed in the Pharmacopoeia, detects the presence or absence of CO2 emitted by bacteria, but because a certain number of bacteria is required to detect CO2, a 7-day incubation period is required to confirm sterility. In other words, both the conventional method and the BacteAlert method have low detection sensitivity, requiring a long incubation period to determine sterility. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2019 / 123692 [Patent Document 2] WO2010 / 082640 Summary of the Invention [Problem to be solved by the invention]

[0006] Digital PCR was developed to quantify nucleic acids. If this method could be used to accurately measure bacterial counts, particularly in patient samples, it would be a valuable new technology for clinical testing of infectious diseases. However, when attempting to accurately quantify bacterial counts using digital PCR, targeting the 16S ribosomal RNA gene, a single bacterium contains multiple 16S ribosomal RNA genes (the number of genes contained in each bacterial species as operon copy number varies). This makes accurate bacterial counts difficult to measure due to DNA fragmentation, resulting in an overestimated bacterial count. Accurate bacterial counts are particularly essential when measuring bacterial counts in clinical samples, making digital PCR difficult to use. Furthermore, there is a need for a method to rapidly determine the presence or absence of many types of bacteria in samples. Therefore, there is a strong demand for technological innovations that enable accurate bacterial counts using digital PCR, even when DNA is fragmented.

[0007] Regarding sterility testing, the conventional microbial testing method described in the Japanese Pharmacopoeia requires more than a week (7-14 days) to determine whether a product is "sterile," which incurs logistics costs before the sterile product (such as pharmaceuticals) is shipped. Furthermore, if a microbial infection is discovered in a sterile product, all sterile products manufactured during that time must be discarded. Furthermore, regenerative medicine products must be used immediately after production, and current sterility testing is insufficient. In other words, there is a demand for rapid sterility testing, both socially and medically. Furthermore, sterility testing would be extremely useful if it could also be performed using digital PCR. [Means for solving the problem]

[0008] The number of 16S ribosomal RNA genes (operon copy number) possessed by a single bacterium varies depending on the bacterial species, and since each bacterium possesses multiple copies (for example, E. coli possesses seven 16S ribosomal RNA genes per bacterium), when DNA is fragmented, the bacterial count measured by digital PCR will be higher than the actual number.

[0009] To solve this problem, the following primer and probe sets were designed and constructed. The target gene was the rpoB gene, which exists only once per bacterium in all bacterial species. The rpoB gene does not contain a conserved region, which is a region of base sequence that is completely conserved across all bacterial species. Therefore, a mixed primer and mixed probe set was designed and created to detect all bacterial species equally well. Especially when used in clinical testing, the gene contains human-derived DNA, and intercalators cannot be used due to the high background, making detection with a probe essential.

[0010] That is, the present invention is as follows. (1)(a) at least one rpoB forward primer selected from the group consisting of SEQ ID NOs: 1 to 19; (b) at least one rpoB reverse primer selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (2)(a) a primer mixture containing at least two rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing at least two rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (3) (a) a primer mixture containing at least five rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing at least five rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (4) (a) a primer mixture containing at least 10 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing at least 10 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (5) (a) a primer mixture containing at least 15 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing at least 12 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (6) (a) a primer mixture containing rpoB forward primers consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing rpoB reverse primers consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (7) (1) a primer pair according to any one of claims 1 to 6; (2) a probe mixture containing rpoB TaqMan probes consisting of SEQ ID NOs: 34 to 43; A PCR reagent comprising: (8) (a) a primer mixture containing equal amounts of at least two rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least two rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (9) (a) a primer mixture containing equal amounts of at least five rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least five rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (10) (a) a primer mixture containing equal amounts of at least 10 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least 10 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (11) (a) a primer mixture containing equal amounts of at least 15 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least 12 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (12) (a) a primer mixture containing equal amounts of rpoB forward primers consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of rpoB reverse primers consisting of SEQ ID NOs: 20 to 33; A primer pair comprising: (13) (i) a primer pair according to any one of (1) to (6) above; (ii) a probe mixture containing equal amounts of rpoB TaqMan probes consisting of SEQ ID NOs: 34 to 43; A PCR reagent comprising: (14) A method for quantifying bacteria in a sample, comprising performing digital PCR using the primer pair described in any one of (1) to (13) above. (15) The quantitative method according to (8) or (14) above, which uses the PCR reagent according to (7) or (13) above. (16) When the above (15) is used for a rapid sterility test, a method for determining whether or not bacteria are growing in a specimen, comprising: (i) extracting nucleic acids from bacteria in a sample before culturing; (ii) culturing the sample; (iii) extracting nucleic acids from the bacteria in the sample after culture; (iv) performing digital PCR on the nucleic acids before and after the culture using the PCR reagent described in (7) or (13) above; (v) determining that live bacteria in the sample have proliferated if the nucleic acid has increased before and after the culture; A method having the following. [Effects of the Invention]

[0011] The accurate measurement of bacterial counts using digital PCR targeting the rpoB gene according to the present invention is particularly useful for measuring bacterial counts in patient samples, i.e., as a new technology for clinical testing. Furthermore, the bacterial counts in patient samples are expected to be useful as novel biomarkers for assessing the severity of infectious diseases and monitoring the effectiveness of treatment. Furthermore, compared to real-time PCR, a major advantage is that there is no need to draw a calibration curve using a quantitative control. Furthermore, the present invention provides a method for rapidly determining the presence or absence of viable bacteria in a sample using digital PCR, i.e., a rapid sterility testing method using digital PCR. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing a schematic representation of the number of bacteria and the test time for the conventional pharmacopoeia method, the pharmacopoeia PacteAlert method, and the test method according to the present invention, relative to the growth curve of a microorganism. [Figure 2] 1 is a flowchart illustrating the implementation of a rapid sterility testing method. DETAILED DESCRIPTION OF THE INVENTION

[0013] The rapid sterility testing method using digital PCR implemented in the present invention will be described below.

[0014] (Preparation of sample-derived nucleic acids) Preparation of nucleic acids from a sample can be carried out by standard methods. It is preferable to prepare nucleic acids from a sample by using a bacterial collection and nucleic acid extraction method that does not cause differences depending on the bacterial species, such as the method used in the Examples described below. When the present invention is carried out as a rapid sterility test, examples of samples include pharmaceuticals, regenerative medicine products, cosmetics, water for daily use, platelet preparations, cell culture medium, and the like.

[0015] (Rapid sterility testing method, Figure 2) The rapid sterility test was carried out as follows. (1) Place the sample into aerobic and anaerobic culture bottles. If necessary, pretreat the sample first (e.g., by membrane filtration). (2) Before incubation (0 hours), the culture medium in the aerobic and anaerobic bottles after the sample was mixed was pelleted and frozen. (3) Cultivate aerobically and anaerobically for a certain period of time (approximately 0.5 to 24 hours) depending on the required speed. (4) Extract nucleic acids (RNA and / or DNA) before culturing (i.e., samples frozen and preserved in (2)). The nucleic acids may be RNA (mainly messenger RNA) only, DNA only, or a mixture of RNA and DNA. Alternatively, by using an adsorbent for PCR inhibitors such as Ampdirect (Shimadzu Corporation), PCR (step (5)) can be performed without going through the nucleic acid extraction step (step (4)). (5) Using digital PCR, confirm whether or not there is an increase in bacterial and fungal nucleic acids (RNA and / or DNA) before and after cultivation.

[0016] (Digital PCR method) Digital PCR was performed with absolute quantification before and after incubation, and no quantitative control was required. Digital PCR was performed as follows.

[0017] (Design and construction of primers and probes targeting the rpoB gene) Primers and probes targeting the rpoB gene were designed. Because the rpoB gene is found only once per bacterium in all bacterial species, it is not affected by DNA fragmentation and allows for accurate measurement of bacterial counts. Furthermore, the rpoB gene does not contain a conserved region, a region of base sequence that is completely conserved across all bacterial species. Therefore, the following mixed primer and probe set was designed and created to detect all bacterial species with approximately the same accuracy.

[0018] The present invention may also use a primer pair containing at least one rpoB forward primer and at least one rpoB reverse primer. The present invention may also use a primer pair containing at least 2 to 15 rpoB forward primers and at least 2 to 12 rpoB reverse primers. Furthermore, a PCR reagent may be prepared containing a probe mixture containing a TaqMan probe in addition to these primer pairs. This reagent enables quantification of bacterial counts using digital PCR. Furthermore, the presence or absence of viable bacterial growth can be determined by using the method of the present invention.

[0019] When two or more types of forward primers are used, the primers can be mixed within a range that can be measured by digital PCR. The mixing ratio should be 10:1 to 1:10, preferably 5:1 to 1:5, more preferably 3:1 to 1:3, and particularly preferably equal amounts.

[0020] When two or more reverse primers are used, the primers can be mixed within a range that can be measured by digital PCR. The mixing ratio should be 10:1 to 1:10, preferably 5:1 to 1:5, more preferably 3:1 to 1:3, and particularly preferably equal amounts.

[0021] [Table 1]

[0022] [Table 2]

[0023] [Table 3] [Example]

[0024] The present invention will be further described below with reference to examples. Unless otherwise specified, digital PCR and various processes were carried out according to various standard methods using known reagents, known instruments, and a commercially available digital PCR device.

[0025] Example 1 Clinical testing was conducted to measure the "bacterial count" using the digital PCR method, using the mixed primers and mixed probes in Tables 1 to 3 that target the rpoB gene. ·Equipment used: QIAcuity One (QUIAGEN) The measurement conditions for this equipment are as follows: Plate type: Nanoplate 26K 24-well Primer-Probe (Qiagen): Assay 3 Primer:rpoB Forward primer 1~19mix rpoB Reverse primer 20-33 mix Probe: rpoB probe 34-43 mix Material: S. aureus (5×10 5 CFU / mL) Reaction solution composition QIAcuity Probe PCR MM 12.5μL rpoB Forward mixed primers 4.0μL rpoB Reverse mixed primers 4.0μL rpoB mixed probes 3.0 μL UCP distilled water 21.5μL DNA 5.0 μL Total 50.0μL Real-time PCR cycle conditions Step Time Temperature(℃) initial heat activation 2min 95 2-step cycling (40 cycles) Denaturation 15s 95 Annealing 1min 55 Cooling down 5min 40

[0026] [Table 4] From the above results, the bacterial count was able to be measured in all clinical specimens.

[0027] Example 2 The flow chart of the rapid test method is shown in Figure 2. Sterility tests were performed using the digital PCR method, and to confirm its usefulness, the samples were cultured under aerobic and anaerobic conditions and the DNA concentration was measured. -Sample inoculation method for sterility testing: 30 mL of sample (eye drops) was filtered and washed three times with 100 mL of washing solution A, and then the membrane filters were placed in culture media and cultured. ·Equipment used: QIAcuity One (QUIAGEN) The measurement conditions for this device were the same as those in Example 1 above. Primer and probe set: The same primer and probe set as in Example 1 above was used.

[0028] (Experimental results) [Table 5]

[0029] The DNA concentrations are shown for the samples cultured under aerobic SCD and anaerobic TGC conditions in Table 4. In Table 4, "aerobic SCD" refers to the results of culturing in aerobic soybean casein digest agar culture bottles, and "anaerobic TGC" refers to the results of culturing in anaerobic thioglycollate agar culture bottles.

[0030] These results confirmed that the new rapid sterility test using digital PCR (the method of the present invention) showed no increase in the number of bacteria per PCR tube even after 24 hours of culture (within the measurement error range (if live bacteria were actually present, the number of bacteria after 24 hours of culture would be several orders of magnitude different)). [Industrial Applicability]

[0031] The primers, probes, primer pairs, primer mixtures, and probe mixtures of the present invention can be used by digital PCR to measure the "bacterial count" in clinical testing for infectious diseases such as sepsis, intrauterine infections, postoperative infections, bacterial meningitis, and bacterial pericarditis. They can also be used in rapid sterility testing of medical products to check for contamination in pharmaceutical manufacturing processes, cosmetic manufacturing processes, surgical instruments, endoscopes, and platelet preparations, as well as in rapid sterility testing of drinking water (commercial products and tap water), pool and hot spring water, cell culture medium (for iPS cells, etc.), and ultrapure water for experiments.

Claims

1. (a) at least one rpoB forward primer selected from the group consisting of SEQ ID NOs: 1-19; (b) at least one rpoB reverse primer selected from the group consisting of SEQ ID NOs: 20-33; A primer pair comprising:

2. (a) a primer mixture comprising at least two rpoB forward primers selected from the group consisting of SEQ ID NOs: 1-19; (b) a primer mixture comprising at least two rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20-33; A primer pair comprising:

3. (a) a primer mixture comprising at least five rpoB forward primers selected from the group consisting of SEQ ID NOs: 1-19; (b) a primer mixture comprising at least five rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20-33; A primer pair comprising:

4. (a) a primer mixture comprising at least 10 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1-19; (b) a primer mixture comprising at least 10 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20-33; A primer pair comprising:

5. (a) a primer mixture comprising at least 15 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1-19; (b) a primer mixture comprising at least 12 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20-33; A primer pair comprising:

6. (a) a primer mixture comprising an rpoB forward primer consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture comprising rpoB reverse primers consisting of SEQ ID NOs: 20 to 33; A primer pair comprising:

7. (1) a primer pair according to any one of claims 1 to 6; (2) a probe mixture comprising rpoB TaqMan probes consisting of SEQ ID NOs: 34 to 43; A reagent for PCR comprising:

8. (a) a primer mixture comprising equal amounts of at least two rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least two rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising:

9. (a) a primer mixture comprising equal amounts of at least five rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least five rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising:

10. (a) a primer mixture comprising equal amounts of at least 10 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least 10 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising:

11. (a) a primer mixture containing equal amounts of at least 15 rpoB forward primers selected from the group consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of at least 12 rpoB reverse primers selected from the group consisting of SEQ ID NOs: 20 to 33; A primer pair comprising:

12. (a) a primer mixture containing equal amounts of rpoB forward primers consisting of SEQ ID NOs: 1 to 19; (b) a primer mixture containing equal amounts of rpoB reverse primers consisting of SEQ ID NOs: 20 to 33; A primer pair comprising:

13. (1) a primer pair according to any one of claims 1 to 6; (2) a probe mixture containing equal amounts of rpoB TaqMan probes consisting of SEQ ID NOs: 34 to 43; A reagent for PCR comprising:

14. A method for quantifying bacteria in a sample, comprising performing digital PCR using the primer pair according to any one of claims 1 to 6.

15. The method for quantification according to claim 14, which uses the PCR reagent according to claim 7.

16. When the above (15) is used for a rapid sterility test, the method for determining the presence or absence of bacterial growth in a specimen comprises: (1) extracting nucleic acid from a bacterial sample before culture; (2) culturing the sample; (3) A step of extracting nucleic acid from the cultured bacteria in the sample (4) performing digital PCR on the nucleic acid before and after the culture using the digital PCR reagent according to claim 7; (5) determining that bacteria in the sample have proliferated when the amount of nucleic acid has increased before and after the culture; The method according to claim 1,

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