Rapid sterility test method

Abstract

The present invention addresses the problem of providing a rapid sterile test method that reduces the obstruction of a human nucleic acid on a nucleic acid amplification reaction of a target nucleic acid. The present invention is a rapid sterility test method for detecting, by a nucleic acid amplification method, the presence of a bacterial nucleic acid contained in a test sample containing a human-derived nucleic acid, the oligonucleotide set used in the nucleic acid amplification method is: (1) one or more probes selected from the group consisting of a probe group PM1, a probe group PM2, and a probe group PM3; and (2) one or more reverse primers in the primer group RM.

Description

Technical Field

[0001] The present invention relates to a rapid sterility test method for detecting bacteria and / or fungal nucleic acids contained in a test sample containing human nucleic acids by a nucleic acid amplification method. Background Art

[0002] In the field of regenerative medicine, cells collected from a subject are sometimes cultured and transplanted into the subject's own or a third party's body. If microorganisms are mixed into the culture medium or culture fluid used in such treatment, there is a possibility that the patient to whom these cells are transplanted may develop an infection due to the microorganisms. Therefore, it is important to confirm the sterility of the sample.

[0003] As methods for confirming the sterility, there are known culture methods and non-culture methods. The culture method is a method for detecting the development of microorganisms using a culture medium in which the target microorganism is grown. However, it has the problem that it takes time from the development of the microorganisms to their detection. On the other hand, in the non-culture method, as a method for detecting the nucleic acid of the target microorganism, polymerase chain reaction (PCR) or real-time PCR are known. Compared with the culture method, the non-culture method can obtain results quickly. However, when the sample contains nucleic acids other than the target microorganism, there is a problem that the nucleic acid hinders the nucleic acid amplification reaction of the target microorganism (non-patent document 1).

[0004] Therefore, it is sought to find and provide a rapid sterility test method that is not easily inhibited by nucleic acid amplification reactions caused by human nucleic acids and can detect a wide range of microorganisms.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: IAN G.WILSON, Inhibition and Facilitation of Nucleic Acid Amplification, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 63, 3741-3751 (1997) Summary of the invention

[0008] 1. Technical issues to be resolved

[0009] When a target nucleic acid contained in a test sample mixed with human cells and / or human-derived nucleic acid is detected by a nucleic acid amplification method, there is a problem that the nucleic acid amplification reaction of the target nucleic acid is hindered. Therefore, the technical problem of the present invention is to provide a rapid sterile test method that reduces the hindrance of the nucleic acid amplification reaction of the target nucleic acid by human-derived nucleic acid.

[0010] (II) Technical solution

[0011] Therefore, in order to solve the above-mentioned technical problems, the inventors of the present application conducted various studies and unexpectedly discovered a rapid sterile test method, which, by using primers and / or probes containing specific base sequences, can reduce the hindrance of human nucleic acid to the nucleic acid amplification reaction of target nucleic acid, thereby completing the present invention.

[0012] That is, the present invention provides the following inventions [1] to

[19] .

[0013] [1] A rapid sterility test method for detecting the presence of bacterial nucleic acid contained in a test sample containing human nucleic acid by a nucleic acid amplification method, characterized in that the oligonucleotide set used in the nucleic acid amplification method is:

[0014] (1) one or more probes selected from probe group PM1, probe group PM2 and probe group PM3, wherein the probe group PM1 comprises the base sequence represented by sequence number 18 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in the probe group PM1 is 12 to 42, the probe group PM2 comprises the base sequence represented by sequence number 27 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in the probe group PM2 is 8 to 42, and the probe group PM3 comprises the base sequence represented by sequence number 30 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in the probe group PM3 is 7 to 42; and

[0015] (2) One or more reverse primers in a primer set RM, wherein the primer set RM comprises the base sequence represented by SEQ ID NO: 4 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the primer set RM is 8 to 30.

[0016] [2] The rapid sterility test method according to [1], characterized in that:

[0017] The probe is one or more probes selected from the base sequences represented by SEQ ID NOs: 21 to 26, SEQ ID NO: 29 or SEQ ID NO: 32, or base sequences obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences,

[0018] The reverse primer is one or more reverse primers selected from the base sequences represented by SEQ ID NOs: 7 to 17, or base sequences in which 1 to 3 bases are inserted, deleted or substituted in each of these base sequences.

[0019] [3] The rapid sterility test method according to [1] or [2], characterized in that the oligonucleotide set further comprises:

[0020] (3) One or more forward primers in primer set FM, wherein the primer set FM comprises the base sequence represented by SEQ ID NO: 1 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the primer set FM is 13 to 50.

[0021] [4] The rapid sterility test method according to [3] is characterized in that the forward primer is one or more forward primers selected from the base sequence represented by sequence number 3 or the base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence.

[0022] [5] The rapid sterility test method according to any one of [1] to [4], wherein the human nucleic acid contained in the test sample is 1 to 1×10 5 ng.

[0023] [6] The rapid sterility test method according to any one of [1] to [5], wherein the number of copies of the detected bacterial nucleic acid is 1 to 100 copies.

[0024] [7] A rapid sterility test method according to any one of [1] to [6], characterized in that the rapid sterility test method uses a nucleic acid extract obtained by adding a nucleic acid degrading enzyme to a test sample that contains at least cells from humans as human nucleic acid and bacteria as bacterial nucleic acid, and extracting nucleic acids contained in human cells and / or bacteria from an enzyme-treated sample from which the nucleic acid degrading enzyme has been removed and / or inactivated.

[0025] [8] The rapid sterility test method according to [7], characterized in that the number of bacteria detected is 1 to 100 CFU.

[0026] [9] A rapid sterility test method for detecting the presence of fungal nucleic acid contained in a test sample containing human nucleic acid by a nucleic acid amplification method, characterized in that the oligonucleotide set used in the nucleic acid amplification method is:

[0027] (1) one or more forward primers in primer set FY1 and primer set FY2, wherein the primer set FY1 comprises the base sequence represented by SEQ ID NO: 33 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in primer set FY1 is 11 to 44, and the primer set FY2 comprises the base sequence represented by SEQ ID NO: 45 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in primer set FY2 is 16 to 44; and

[0028] (2) One or more reverse primers in a primer set RY, wherein the primer set RY comprises the base sequence represented by SEQ ID NO: 51 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the primer set RY is 17 to 44.

[0029]

[10] The rapid sterility test method according to [9], characterized in that:

[0030] The forward primer is one or more forward primers selected from the base sequences represented by SEQ ID NOs: 37 to 44, or 46 to 48, or base sequences obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences,

[0031] The reverse primer is one or more reverse primers selected from the base sequences represented by SEQ ID NOs: 52 to 55, or base sequences in which 1 to 3 bases are inserted, deleted or substituted in each of these base sequences.

[0032]

[11] The rapid sterility test method according to [9] or

[10] , characterized in that the oligonucleotide set further comprises:

[0033] (3) One or more probes in a probe set PY, wherein the probe set PY comprises the base sequence represented by SEQ ID NO: 56 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the probe set PY is 17 to 50.

[0034]

[12] The rapid sterility test method according to

[11] is characterized in that the probe is one or more probes selected from the base sequence represented by sequence number 58 or the base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence.

[0035]

[13] The rapid sterility test method according to any one of [9] to

[12] , characterized in that the human nucleic acid contained in the test sample is 1 to 1×10 5 ng.

[0036]

[14] The rapid sterility test method according to any one of [9] to

[13] , characterized in that the number of copies of the detected fungal nucleic acid is 1 to 100 copies.

[0037]

[15] A rapid aseptic test method according to any one of [9] to

[14] , characterized in that the rapid aseptic test method uses a nucleic acid extract obtained by adding a nucleic acid degrading enzyme to a test sample that contains at least cells from humans as human nucleic acid and fungi as fungal nucleic acid, and extracting nucleic acids from human cells and / or fungi from an enzyme-treated sample from which the nucleic acid degrading enzyme has been removed and / or inactivated.

[0038]

[16] The rapid sterility test method according to

[15] is characterized in that the number of fungi detected is 1 to 100 CFU.

[0039]

[17] The rapid sterility test method according to any one of [7], [8],

[15] and

[16] , characterized in that the number of cells derived from humans is 1 to 1×10 7 cells.

[0040]

[18] The rapid sterility test method according to any one of [1] to

[17] , characterized in that the nucleic acid amplification method is a PCR method or a real-time PCR method.

[0041]

[19] A real-time PCR kit, wherein the kit is used for the rapid sterility test method described in any one of [1] to

[18] .

[0042] (III) Beneficial effects

[0043] According to the present invention, by selecting primers and / or probes containing specific base sequences, it is possible to reduce the inhibition of the nucleic acid amplification reaction of the target nucleic acid by the human-derived nucleic acid, and thus the present invention is extremely excellent as a rapid sterility test method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 A calculation formula for determining the copy concentration of a standard DNA solution is shown.

[0045] Figure 2 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 1 is shown (human nucleic acid concentration: 1000 ng).

[0046] Figure 3 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 2 is shown (human nucleic acid concentration: 1000 ng).

[0047] Figure 4An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 2 is shown (human nucleic acid concentration: 100 ng).

[0048] Figure 5 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 2 is shown (human nucleic acid concentration: 10 ng).

[0049] Figure 6 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 3 is shown (human nucleic acid concentration: 1000 ng).

[0050] Figure 7 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 3 is shown (human nucleic acid concentration: 100 ng).

[0051] Figure 8 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 3 is shown (human nucleic acid concentration: 10 ng).

[0052] Fig. 9 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 4 is shown (human nucleic acid concentration: 1000 ng).

[0053] Fig.10 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 5 is shown (human nucleic acid concentration: 1000 ng).

[0054] Fig.11 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 6 is shown (human nucleic acid concentration: 1000 ng).

[0055] Fig.12 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 6 is shown (human nucleic acid concentration: 10 ng).

[0056] Fig.13 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 7 is shown (human nucleic acid concentration: 1000 ng).

[0057] Fig.14 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 8 is shown (human nucleic acid concentration: 1000 ng).

[0058] Fig.15 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 9 is shown (human nucleic acid concentration: 1000 ng).

[0059] Fig.16 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 9 is shown (human nucleic acid concentration: 10 ng).

[0060] Fig.17 An amplification curve of a nucleic acid amplification reaction using the oligonucleotide set 10 is shown (human nucleic acid concentration: 1000 ng).

[0061] Fig.18 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 11 is shown (human nucleic acid concentration: 1000 ng).

[0062] Fig.19 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 12 is shown (human nucleic acid concentration: 1000 ng).

[0063] Fig. 20 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 13 is shown (human nucleic acid concentration: 1000 ng).

[0064] Fig.21 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 13 is shown (human nucleic acid concentration: 100 ng).

[0065] Fig. 22 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 13 is shown (human nucleic acid concentration: 10 ng).

[0066] Fig.23 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 14 is shown (human nucleic acid concentration: 1000 ng).

[0067] Fig.24 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 15 is shown (human nucleic acid concentration: 1000 ng).

[0068] Fig.25 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 15 is shown (human nucleic acid concentration: 10 ng).

[0069] Fig.26 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 16 is shown (human nucleic acid concentration: 1000 ng).

[0070] Fig. 27 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 17 is shown (human nucleic acid concentration: 1000 ng).

[0071] Fig.28 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 18 is shown (human nucleic acid concentration: 1000 ng).

[0072] Fig.29An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 19 is shown (human nucleic acid concentration: 1000 ng).

[0073] Fig.30 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 20 is shown (human nucleic acid concentration: 1000 ng).

[0074] Fig.31 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 21 is shown (human nucleic acid concentration: 1000 ng).

[0075] Fig.32 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 21 is shown (human nucleic acid concentration: 10 ng).

[0076] Fig.33 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 22 is shown (human nucleic acid concentration: 1000 ng).

[0077] Fig.34 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 23 is shown (human nucleic acid concentration: 1000 ng).

[0078] Fig.35 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 23 is shown (human nucleic acid concentration: 10 ng).

[0079] Fig.36 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 24 is shown (human nucleic acid concentration: 1000 ng).

[0080] Fig.37 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 25 is shown (human nucleic acid concentration: 1000 ng).

[0081] Fig.38 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 25 is shown (human nucleic acid concentration: 100 ng).

[0082] Fig.39 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 25 is shown (human nucleic acid concentration: 10 ng).

[0083] Fig.40 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 26 is shown (human nucleic acid concentration: 1000 ng).

[0084] Fig.41 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 26 is shown (human nucleic acid concentration: 10 ng).

[0085] Fig.42 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 27 is shown (human nucleic acid concentration: 1000 ng).

[0086] Fig.43 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 27 is shown (human nucleic acid concentration: 100 ng).

[0087] Fig.44 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 27 is shown (human nucleic acid concentration: 10 ng).

[0088] Fig.45 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 28 is shown (human nucleic acid concentration: 1000 ng).

[0089] Fig.46 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 28 is shown (human nucleic acid concentration: 100 ng).

[0090] Fig.47 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 28 is shown (human nucleic acid concentration: 10 ng).

[0091] Fig.48 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 29 is shown (human nucleic acid concentration: 1000 ng).

[0092] Fig.49 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 30 is shown (human nucleic acid concentration: 1000 ng).

[0093] Fig.50 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 30 is shown (human nucleic acid concentration: 10 ng).

[0094] Fig.51 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 31 is shown (human nucleic acid concentration: 1000 ng).

[0095] Fig.52 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 32 ​​is shown (human nucleic acid concentration: 1000 ng).

[0096] Fig.53 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 32 ​​is shown (human nucleic acid concentration: 100 ng).

[0097] Fig.54An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 32 ​​is shown (human nucleic acid concentration: 10 ng).

[0098] Fig.55 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 33 is shown (human nucleic acid concentration: 1000 ng).

[0099] Fig.56 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 33 is shown (human nucleic acid concentration: 100 ng).

[0100] Fig.57 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 33 is shown (human nucleic acid concentration: 10 ng).

[0101] Fig.58 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 34 is shown (human nucleic acid concentration: 1000 ng).

[0102] Fig.59 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 34 is shown (human nucleic acid concentration: 100 ng).

[0103] Fig.60 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 34 is shown (human nucleic acid concentration: 10 ng).

[0104] Fig.61 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 35 is shown (human nucleic acid concentration: 1000 ng).

[0105] Fig.62 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 36 is shown (human nucleic acid concentration: 1000 ng).

[0106] Fig.63 An amplification curve of a nucleic acid amplification reaction using oligonucleotide set 36 is shown (human nucleic acid concentration: 10 ng).

[0107] Fig.64 (1) An amplification curve of a bacterial nucleic acid amplification reaction using oligonucleotide set 20 is shown.

[0108] Fig.65 (2) An amplification curve of a bacterial nucleic acid amplification reaction using oligonucleotide set 20 is shown.

[0109] Fig.66 (3) An amplification curve of a bacterial nucleic acid amplification reaction using oligonucleotide set 20 is shown.

[0110] Fig.67(4) An amplification curve of a bacterial nucleic acid amplification reaction using oligonucleotide set 20 is shown.

[0111] Fig.68 (5) shows the amplification curve of the bacterial nucleic acid amplification reaction using oligonucleotide set 20.

[0112] Fig.69 (6) shows the amplification curve of the bacterial nucleic acid amplification reaction using oligonucleotide set 20.

[0113] Fig.70 (7) shows the amplification curve of the bacterial nucleic acid amplification reaction using oligonucleotide set 20.

[0114] Fig.71 (8) shows the amplification curve of the bacterial nucleic acid amplification reaction using oligonucleotide set 20.

[0115] Fig.72 (1) Amplification curve of a nucleic acid amplification reaction of fungi using oligonucleotide set 21 is shown.

[0116] Fig.73 (2) Amplification curve of a nucleic acid amplification reaction of fungi using oligonucleotide set 21 is shown.

[0117] Fig.74 (3) shows the amplification curve of the nucleic acid amplification reaction of fungi using oligonucleotide set 21.

[0118] Fig.75 (4) shows the amplification curve of the nucleic acid amplification reaction of fungi using oligonucleotide set 21. DETAILED DESCRIPTION

[0119] The present invention is a rapid sterile test method for detecting the presence of a target nucleic acid contained in a test sample containing a human-derived nucleic acid by a nucleic acid amplification reaction, and uses an oligonucleotide set in the nucleic acid amplification reaction.

[0120] The target nucleic acid of the present invention refers to all or part of the DNA or RNA of the target microorganism. When the target microorganism is a bacterium, it refers to bacterial nucleic acid; when the target microorganism is a fungus, it refers to fungal nucleic acid.

[0121] The copy number of the present invention refers to the total number of regions (nucleic acid amplification regions) specified by forward primers and reverse primers as the object of nucleic acid amplification reaction. Due to the presence of repetitive sequences in nucleic acids, multiple nucleic acid amplification regions may sometimes exist in each genome. For example, when the nucleic acid amplification region of the target microorganism is present in a repetitive sequence and there are 10 repetitive sequences in each genome, the copy number of the nucleic acid is 10 copies. Further, when the test sample contains 10 such genomes, the copy number of the target microorganism of the test sample is 100 copies (10 copies × 10 genomes). In order to ensure the performance of the rapid sterility test, it is preferred to be able to detect a smaller copy number, so in the nucleic acid amplification reaction unit, the copy number is preferably 1 to 1000 copies, more preferably 1 to 100 copies, and further preferably 1 to 10 copies.

[0122] Here, the nucleic acid amplification reaction unit refers to a unit of nucleic acid amplification reaction, and for example, in the real-time PCR method, it refers to each well.

[0123] In addition, the target microorganism itself can also be used as the target nucleic acid. In order to ensure the performance of the rapid sterility test, it is preferred to be able to detect a smaller number, so in the nucleic acid amplification reaction unit, as the target microorganism, it is preferred to be able to detect 1 to 100 colony forming units (CFU, colony forming units) of the target microorganism, more preferably 1 to 50 CFU of the target microorganism, and further preferably 1 to 10 CFU of the target microorganism.

[0124] In addition, in a sterility test performed by a culture method according to the 18th edition of the Japanese Pharmacopoeia (JP), it is required to be able to detect at least 100 CFU of a target microorganism contained in a test sample of the sterility test.

[0125] The target microorganism refers to a microorganism contained or possibly contained in a test sample, and examples of the microorganism include target bacteria such as bacteria and target fungi such as fungi.

[0126] In particular, in the Japanese Pharmacopoeia, which is a standard method for sterility testing, Clostridium sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis are listed as bacteria, and Aspergillus brasiliensis and Candida albicans are listed as fungi. From the perspective of compliance with the Japanese Pharmacopoeia, it is preferred that these bacteria and fungi can be detected. In addition, in the European Pharmacopoeia 10th Edition, the same species as those in the Japanese Pharmacopoeia are listed.

[0127] Furthermore, in order to confirm that the sample is not contaminated by microorganisms, it is further preferred to be able to detect microorganisms other than the above. For example, microorganisms isolated from indoor environments such as product manufacturing rooms, inspection rooms, and laboratories (walls and floors of buildings, surfaces of equipment and machines, indoor air, raw materials, equipment, water, etc.), and microorganisms that are pathogenic to humans can be listed.

[0128] The oligonucleotide set of the present invention refers to a primer set or a primer and probe set. The primer set is composed of a combination of a forward primer and a reverse primer. The primer and probe set is composed of a forward primer and a probe, a reverse primer and a probe, or a combination of a forward primer, a reverse primer and a probe.

[0129] When bacteria are targeted, the forward primer, reverse primer, and probe that can be used are the following.

[0130] As the probe for bacteria, the probe set PM can be used. As the probe set PM, one or more probes of the probe set PM1, the probe set PM2, and the probe set PM3 can be used.

[0131] As the probe set PM1, it is preferred to include the base sequence represented by sequence number 18, more preferably the base sequence represented by sequence number 19, further preferably the base sequence represented by sequence number 20, and further preferably the base sequence represented by sequence number 21. The base sequences represented by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in the probe set PM1 is preferably 12 to 42, more preferably 17 to 39, further preferably 20 to 26, and further preferably 23 to 26. Specifically, the probe set PM1 is preferably a base sequence represented by sequence numbers 18 to 25 or a probe in which 1 to 3 bases are inserted, deleted or replaced in each of these base sequences.

[0132] As probe set PM2, it is preferred to include the base sequence represented by sequence number 27, more preferably the base sequence represented by sequence number 28, and further preferably the base sequence represented by sequence number 29. The base sequences represented by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in probe set PM2 is preferably 8 to 42, more preferably 16 to 39, and further preferably 19 to 22. Specifically, probe set PM2 is preferably a base sequence represented by sequence numbers 23 to 25, or sequence numbers 27 to 29, or a probe in which 1 to 3 bases are inserted, deleted or replaced in each of these base sequences.

[0133] As the probe set PM3, it is preferred to include the base sequence represented by sequence number 30, more preferably the base sequence represented by sequence number 31, and further preferably the base sequence represented by sequence number 32. The base sequences represented by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in the probe set PM3 is preferably 7 to 42, more preferably 16 to 39, and further preferably 19 to 22. Specifically, the probe set PM3 is preferably a base sequence represented by sequence number 21, sequence number 22, sequence number 24, sequence number 26 or sequence number 30 to sequence number 32, or a probe in which 1 to 3 bases are inserted, deleted or replaced in each of these base sequences.

[0134] As the reverse primer for bacteria, primer set RM can be used.

[0135] As the primer set RM, it is preferred to include the base sequence represented by sequence number 4, more preferably the base sequence represented by sequence number 5, further preferably the base sequence represented by sequence number 6, and further preferably the base sequence represented by sequence number 7. The base sequences shown by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in the primer set RM is preferably 8 to 30, more preferably 13 to 27, further preferably 16 to 23, and further preferably 19 to 22. Specifically, the primer set RM is preferably a base sequence represented by sequence numbers 4 to 17 or a primer in which 1 to 3 bases are inserted, deleted or replaced in each of these base sequences, and is more preferably a base sequence represented by sequence number 7, sequence number 8 or sequence number 10 to 17 or a primer in which 1 to 3 bases are inserted, deleted or replaced in each of these base sequences.

[0136] From the perspective of reducing the hindrance of human nucleic acid to the nucleic acid amplification reaction of target nucleic acid, the combination of probe group PM and primer group RM is preferably: a probe comprising sequence number 18 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences and having a base number of 12 to 42, a probe comprising sequence number 27 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences and having a base number of 8 to 42, or a probe comprising sequence number 30 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences and having a base number of 7 to 42, and a combination of primers comprising the base sequence represented by sequence number 4 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having a base number of 8 to 30. More preferred is a combination of a base sequence represented by sequence numbers 21 to 26, 29 or 32, or a probe for inserting, deleting or replacing 1 to 3 bases in each of these base sequences, and a base sequence represented by sequence numbers 7 to 17, or a primer for inserting, deleting or replacing 1 to 3 bases in each of these base sequences. Even more preferred is a combination of a base sequence represented by sequence number 21, or a probe for inserting, deleting or replacing 1 to 3 bases in the base sequence, and a base sequence represented by sequence number 7, or a primer for inserting, deleting or replacing 1 to 3 bases in the base sequence.

[0137] Furthermore, from the perspective of comprehensive detection of target bacteria, it is preferred to use a combination of probes of base sequences represented by SEQ ID NOs: 21 and 22 and primers represented by base sequences represented by SEQ ID NOs: 7, 8, and 9. These probes and primers may be probes and primers in which 1 to 3 bases are inserted, deleted, or substituted in each base sequence.

[0138] In addition, the forward primer for bacteria can be appropriately selected according to the target bacteria to be detected. As the forward primer for bacteria, the primer set FM can be used according to the length of the base sequence of the product of the reaction in the nucleic acid amplification reaction.

[0139] As primer set FM, it is preferred to include the base sequence represented by sequence number 1, more preferably the base sequence represented by sequence number 2, and further preferably the base sequence represented by sequence number 3. The base sequences represented by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in primer set FM is preferably 13 to 50, more preferably 16 to 40, and further preferably 19 to 22. Specifically, primer set FM preferably includes the base sequences represented by sequence numbers 1 to 3 or primers in which 1 to 3 bases are inserted, deleted or replaced in each of these base sequences.

[0140] Furthermore, from the perspective of reducing the hindrance of human nucleic acid to the nucleic acid amplification reaction of target nucleic acid, the combination of the forward primer and primer set RM for bacteria is preferably: a primer comprising the base sequence represented by sequence number 1 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 13 to 50 bases, and a primer comprising the base sequence represented by sequence number 4 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 8 to 30 bases. More preferred is a combination of a primer comprising a base sequence represented by sequence number 2 or sequence number 3, or a primer comprising inserting, deleting or replacing 1 to 3 bases in each of these base sequences, and a primer comprising a base sequence represented by sequence number 7 to sequence number 17, or a primer comprising inserting, deleting or replacing 1 to 3 bases in each of these base sequences. Further preferred is a combination of a primer comprising a base sequence represented by sequence number 3, or a primer comprising inserting, deleting or replacing 1 to 3 bases in this base sequence, and a primer comprising a base sequence represented by sequence number 7, or a primer comprising inserting, deleting or replacing 1 to 3 bases in this base sequence.

[0141] Furthermore, from the perspective of comprehensive detection of target bacteria, it is more preferable to use a combination of the primer represented by SEQ ID NO: 3 and the primers represented by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. These primers may also be primers in which 1 to 3 bases are inserted, deleted, or substituted in each base sequence.

[0142] In addition, from the perspective of reducing the hindrance of the nucleic acid amplification reaction of the target nucleic acid by the human nucleic acid, the combination of the forward primer, probe set PM and primer set RM for bacteria is preferably: a primer comprising the base sequence represented by sequence number 1 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having a base number of 13 to 50, and a primer comprising sequence number 18 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences and having a base number of 12 to 42. A probe comprising sequence number 27 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences and having 8 to 42 bases, or a probe comprising sequence number 30 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences and having 7 to 42 bases, and a primer comprising the base sequence represented by sequence number 4 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 8 to 30 bases. More preferably, it is: a base sequence represented by sequence number 2 or sequence number 3, or a primer that inserts, deletes or replaces 1 to 3 bases in each of these base sequences, and a base sequence represented by sequence number 21 to sequence number 26, sequence number 29, or sequence number 32, or a probe that inserts, deletes or replaces 1 to 3 bases in each of these base sequences, and a base sequence represented by sequence number 7 to sequence number 17, or a primer that inserts, deletes or replaces 1 to 3 bases in each of these base sequences; further preferably, it is: a base sequence represented by sequence number 3, or a primer that inserts, deletes or replaces 1 to 3 bases in this base sequence, and a base sequence represented by sequence number 21, or a probe that inserts, deletes or replaces 1 to 3 bases in this base sequence, and a base sequence represented by sequence number 7, or a primer that inserts, deletes or replaces 1 to 3 bases in this base sequence.

[0143] Furthermore, from the perspective of comprehensive detection of target bacteria, it is preferred to use a combination of the primer represented by SEQ ID NO: 3, the probe represented by SEQ ID NO: 21 and SEQ ID NO: 22, and the primer represented by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. These primers and probes may be primers and probes in which 1 to 3 bases are inserted, deleted, or substituted in each base sequence.

[0144] When fungi are targeted, the following forward primers, reverse primers, and probes can be used.

[0145] As the forward primer for fungi, primer set FY can be used. As primer set FY, one or more primers of primer set FY1 and primer set FY2 can be used.

[0146] As primer set FY1, it is preferred to include the base sequence represented by sequence number 33, more preferably the base sequence represented by sequence number 35, further preferably the base sequence represented by sequence number 36, and further preferably the base sequence represented by sequence number 37. The base sequences represented by these sequence numbers may be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in primer set FY1 is preferably 11 to 44, more preferably 15 to 41, further preferably 16 to 26, and further preferably 19 to 22. Specifically, primer set FY1 is preferably a primer having a base sequence represented by sequence numbers 33 to 48 or a primer having 1 to 3 bases inserted, deleted or replaced in each of these base sequences, and more preferably a primer having a base sequence represented by sequence numbers 33 to 40, sequence number 42, sequence number 43, or sequence numbers 45 to 48 or a primer having 1 to 3 bases inserted, deleted or replaced in each of these base sequences.

[0147] As primer set FY2, it is preferred to include the base sequence represented by sequence number 45, and more preferably the base sequence represented by sequence number 46. The base sequences represented by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in primer set FY2 is preferably 16 to 44, more preferably 19 to 40, further preferably 19 to 37, and further preferably 19 to 22. Specifically, primer set FY2 is preferably a primer having base sequences represented by sequence numbers 45 to 48 or having 1 to 3 bases inserted, deleted or replaced in each of these base sequences.

[0148] As the reverse primer for fungi, primer set RY can be used.

[0149] As the primer set RY, it is preferred to include the base sequence represented by sequence number 51, and more preferably the base sequence represented by sequence number 52. The base sequences represented by these sequence numbers may be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in the primer set RY is preferably 17 to 44, more preferably 18 to 39, further preferably 18 to 36, and further preferably 21 to 24. Specifically, the primer set RY is preferably a primer in which the base sequences represented by sequence numbers 51 to 55 or 1 to 3 bases are inserted, deleted or replaced in each of these base sequences.

[0150] From the perspective of reducing the hindrance of the nucleic acid amplification reaction of the target nucleic acid by the human nucleic acid, the combination of primer set FY and primer set RY is preferably: a primer comprising the base sequence represented by sequence number 33 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 11 to 44 bases, or a primer comprising the base sequence represented by sequence number 45 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 16 to 44 bases, and a primer comprising the base sequence represented by sequence number 51 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 16 to 44 bases. A combination of primers with sequence numbers 17 to 44; more preferably, a combination of primers with base sequences represented by sequence numbers 37 to 44, or sequence numbers 46 to 48, or primers with 1 to 3 bases inserted, deleted or substituted in each of these base sequences, and a combination of primers with base sequences represented by sequence numbers 52 to 55, or primers with 1 to 3 bases inserted, deleted or substituted in each of these base sequences; further preferably, a combination of a primer with a base sequence represented by sequence number 37, or a primer with 1 to 3 bases inserted, deleted or substituted in the base sequence, and a primer with a base sequence represented by sequence number 52, or a primer with 1 to 3 bases inserted, deleted or substituted in the base sequence.

[0151] Furthermore, from the viewpoint of comprehensive detection of the target fungus, one or more primer sets FY and one or more primer sets RY may be combined.

[0152] In addition, the probe for fungi can be appropriately selected according to the target bacteria to be detected. As the probe for fungi, the probe set PY can be used.

[0153] As the probe set PY, it is preferred to include the base sequence represented by sequence number 56, more preferably the base sequence represented by sequence number 57, and further preferably the base sequence represented by sequence number 58. The base sequences represented by these sequence numbers can be base sequences obtained by inserting, deleting or replacing 1 to 3 bases. The number of bases in the probe set PY is preferably 17 to 50, more preferably 20 to 40, and further preferably 23 to 26. Specifically, the probe set PY is preferably a probe having base sequences represented by sequence numbers 56 to 58 or having 1 to 3 bases inserted, deleted or replaced in each of these base sequences.

[0154] In addition, from the perspective of reducing the hindrance of the nucleic acid amplification reaction of the target nucleic acid by the human nucleic acid, the combination of the primer set FY, the probe for fungi and the primer set RM is preferably: a primer comprising the base sequence represented by sequence number 33 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 11 to 44 bases, or a primer comprising the base sequence represented by sequence number 45 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 16 to 44 bases, and a probe comprising the base sequence represented by sequence number 56 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 17 to 50 bases, and a primer comprising the base sequence represented by sequence number 51 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence and having 17 to 44 bases. a combination of a base sequence represented by sequence numbers 37 to 44 or sequence numbers 46 to 48, or a primer for inserting, deleting or replacing 1 to 3 bases in each of these base sequences, and a probe for inserting, deleting or replacing 1 to 3 bases in each of these base sequences, and a base sequence represented by sequence numbers 57 or 58, or a primer for inserting, deleting or replacing 1 to 3 bases in each of these base sequences, and a base sequence represented by sequence numbers 52 to 55, or a primer for inserting, deleting or replacing 1 to 3 bases in each of these base sequences; a further preferred combination is a base sequence represented by sequence number 37, or a primer for inserting, deleting or replacing 1 to 3 bases in the base sequence, and a base sequence represented by sequence number 58, or a primer for inserting, deleting or replacing 1 to 3 bases in the base sequence, and a base sequence represented by sequence number 52, or a primer for inserting, deleting or replacing 1 to 3 bases in the base sequence.

[0155] Furthermore, from the viewpoint of comprehensive detection of the target fungus, one or more primer sets FY, one or more probes for fungi, and one or more primer sets RM may be combined.

[0156] The human nucleic acid of the present invention refers to a nucleic acid derived from a human cell. Examples of human-derived cells include cells collected from a human organism, cells modified from the cells (including cells processed from cell lines, iPS cells, ES cells, etc., but not limited thereto), and cells obtained by culturing the above cells, but not limited thereto.

[0157] The concentration of human nucleic acid can be measured by existing methods. For example, nucleic acid from human cells can be extracted and measured using the ultra-micro spectrophotometer NanoDrop (Thermo Fisher Scientific). From the perspective of reducing the hindrance of human nucleic acid to the nucleic acid amplification reaction of the target nucleic acid, the concentration of human nucleic acid is preferably 1 to 1×10 5 ng, more preferably 10 to 1×10 4 ng, more preferably 100 to 5000 ng, and even more preferably 1000 to 2500 ng. From the perspective of reducing the hindrance of the human nucleic acid to the nucleic acid amplification reaction of the target nucleic acid, as the ratio of the target nucleic acid to the human nucleic acid, the human nucleic acid is preferably 10 to 1000 ng relative to 1000 copies of the target nucleic acid, more preferably 10 to 1000 ng relative to 100 copies of the target nucleic acid, and even more preferably 10 to 1000 ng relative to 10 copies of the target nucleic acid.

[0158] In addition, human-derived cells themselves can also be used as human-derived nucleic acids. From the perspective of reducing the hindrance of the human-derived nucleic acid to the nucleic acid amplification reaction of the target nucleic acid, the number of human-derived cells in the nucleic acid amplification reaction unit is preferably 1 to 1×10 7 cells, more preferably 1000 to 1×10 6 cells, more preferably 1×10 4 ~5×10 5 cells, more preferably 1×10 5 ~2.5×10 5 cells.

[0159] The test sample of the present invention refers to a sample that is the object of the sterility test, and examples thereof include water, buffer, physiological saline, biological samples (blood, urine, saliva, cerebrospinal fluid, etc.), cell culture fluid, culture supernatant, culture medium, etc. It is expected to contain cells from humans and / or human nucleic acids. The test sample can be used directly, but in order to effectively recover the target microorganisms in the test sample, a sample from which the microorganisms are collected (bacteria collection sample) can also be used, and in order to improve the detection sensitivity of the rapid sterility test, a sample from which the target nucleic acid is extracted (nucleic acid extraction sample) can also be used. In addition, when the test sample contains at least cells from humans and target microorganisms, after being treated with a nucleic acid degrading enzyme, a sample from which nucleic acid has been extracted (enzyme-treated sample) can also be used.

[0160] The rapid sterility test method of the present invention is a method for detecting a target nucleic acid contained in a test sample using a nucleic acid amplification method, etc. If the target nucleic acid is not detected by the rapid sterility test method, it can be determined that the target microorganism is not present in the test sample provided in the test.

[0161] The nucleic acid amplification method of the present invention can be an existing method, and PCR method, real-time PCR method, etc. can be listed. In particular, from the perspective of being able to confirm the amplification status in real time, the real-time PCR method is preferred. In the real-time PCR method, the chimeric method and the probe method can be listed. As chimeric, SYBR Green, etc. are known. In addition, as the probe method, the TaqMan probe method, etc. are known, and from the perspective of specificity to the target nucleic acid, the TaqMan probe method is preferably used.

[0162] The reaction conditions of the nucleic acid amplification method may be ordinary thermal denaturation, hybridization, and extension reactions, and the detailed conditions may be appropriately changed depending on the enzyme or oligonucleotide set used.

[0163] As a method for preparing a sample for collecting bacteria, for example, a method of adding protein and / or a hydrophobic carrier to a sample to collect bacteria can be used. More specifically, the methods described in PCT / JP2018 / 016676 and PCT / JP2019 / 41658 can be used. In summary, bacteria are collected by adding a protein such as BSA alone or further adding a hydrophobic carrier such as latex particles, and recovering the precipitate by centrifugation. In this way, the target microorganisms can be effectively recovered.

[0164] As a method for preparing nucleic acid extraction samples, an existing method may be used. Nucleic acid extraction is mainly divided into two steps: lysis and purification.

[0165] Examples of the method for lysing bacteria include physical disruption such as bead milling or enzyme treatment using an enzyme that lyses each microorganism, but are not limited thereto. Physical disruption is preferred because it can disrupt bacterial cells regardless of bacterial species.

[0166] Examples of the purification method include, but are not limited to, a magnetic bead method, a purification column method, and a phenol / chloroform method.

[0167] The equipment used for lysis and purification is preferably one that does not contain nucleic acid, and more preferably one that is easy to handle. For example, as a purification method, QIAamp UCP DNA MicroKit using a purification column method can be cited.

[0168] As a method for preparing an enzyme-treated sample, it can be obtained by the following method: adding a nucleic acid degrading enzyme to a test sample containing at least cells from humans and target microorganisms, removing the nucleic acid degrading enzyme and / or inactivating the nucleic acid degrading enzyme, and then extracting nucleic acid. Although the nucleic acid degrading enzyme cannot decompose nucleic acids present in cells, it can decompose free nucleic acids. Free nucleic acids refer to nucleic acids present outside cells, for example, nucleic acids leaked outside cells can be listed, but are not limited to this. As a result, only target microorganisms with proliferative properties can be detected by nucleic acid amplification method.

[0169] The nucleic acid degrading enzyme is not particularly specified, but Turbo Nuclease (endonuclease) is preferred. The method for extracting nucleic acid used in the enzyme-treated sample is not particularly specified, and a known method may be used.

[0170] The present invention can be made into a nucleic acid amplification kit comprising an oligonucleotide set. Examples of nucleic acid amplification kits include, but are not limited to, PCR kits and real-time PCR kits. In addition, specifically, in a rapid sterility test for bacteria, it is a nucleic acid amplification kit comprising the primer set FM and primer set RM, or primer set FM, primer set RM and probe set PM, and in a rapid sterility test for fungi, it is a nucleic acid amplification kit comprising the primer set FY and primer set RY, or primer set FY, primer set RY and probe set PY.

[0171] In addition, the kit may also include a bacteria collection kit, a reagent for destroying cells other than the target microorganism, a nucleic acid extraction kit, a reagent for decomposing free nucleic acids, a protocol for conducting the test, etc. The kit can be used for a rapid sterility test to quickly detect the target nucleic acid contained in the test sample.

[0172] Example

[0173] Next, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these Examples in any way.

[0174] Example 1

[0175] [Oligonucleotide Set]

[0176] The oligonucleotide sets used in the Examples are shown in Table 1.

[0177] The primers and probes used in the examples were designed using bacterial and fungal sequence databases, and the primers and probes targeted the region encoding 16S ribosomal RNA for bacteria and the region encoding 18S ribosomal RNA for fungi, respectively.

[0178] The oligonucleotide set formed by combining the designed primers and probes is compared with the region encoding the 16S or 18S ribosomal RNA of the target microorganism to confirm whether it is a combination in which the forward primer, probe, and reverse primer are all 100% identical. For example, when a portion of the sequence of a strain in a certain bacterial species is 100% identical to the sequences of sequence number 3, sequence number 7, and sequence number 21, respectively, it is determined that the combination of the forward primer, probe, and reverse primer is 100% identical to the bacterial species, and it is determined that the bacterial species can be detected.

[0179] As an example, part of the results of using oligonucleotide set 20 for bacteria and oligonucleotide set 21 for fungi are shown. In addition, the results show the number of species contained in the genus name of microorganisms that are of high clinical importance or that may be detected from the preparation environment among the species that conform to the database. For bacteria, 815 species can be detected (Table 2). These include Clostridium sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. For fungi, 506 species can be detected (Table 3). These include 2 species recorded in the Japanese Pharmacopoeia (Aspergillus brasiliensis and Candida albicans).

[0180] [Table 1]

[0181]

[0182] [Table 2]

[0183]

[0184] [Table 3]

[0185]

[0186] [Human nucleic acid]

[0187] Human nucleic acid was extracted from Jurkat cells derived from human leukemia T cells. 0.5 mL of 2.5 × 10 6 The solution obtained by extracting a Jurkat cell suspension of 10 cells / mL (final 100 μL). The nucleic acid concentration contained in the obtained human nucleic acid extract was measured by an ultra-micro spectrophotometer NanoDrop (Thermo Fisher Scientific). The human nucleic acid extract was prepared by appropriately diluting the human nucleic acid extract to a specified concentration.

[0188] As an example, the concentration of human nucleic acid extract used in the bacterial test was 71.5 ng / μL. Therefore, 1.75×10 5 cells, and 100ng of human nucleic acid is 1.75×10 4 cells, and 10ng of human nucleic acid is 1.75×10 3 cells.

[0189] [Target nucleic acid]

[0190] For the target nucleic acid, the bacterial nucleic acid was extracted from Staphylococcus aureus ATCC 29213 to obtain a target nucleic acid extract, and the fungal nucleic acid was extracted from Candida albicans ATCC 10231 to obtain a target nucleic acid extract.

[0191] Next, the copy numbers of these target nucleic acid extracts were calculated using a standard curve obtained by measuring the copy number of a standard DNA known by real-time PCR. 3 Copy, 10 4 Copy, 10 5 Copy, 10 6 The standard DNA solution prepared in a copy manner and the target nucleic acid extract diluted 100 times were measured (template in Table 4). The real-time PCR conditions are shown in Tables 4 and 5. The vertical axis is set to the Cq value, and the horizontal axis is set to the copy number (displaying the log with a base of 10). The standard curve is prepared using the 4 measured values ​​of the standard DNA, and the copy number of the target nucleic acid is calculated from the Cq value. The target nucleic acid extract is appropriately diluted to form a specified copy number.

[0192] As an example, Staphylococcus aureus is shown. The results of real-time PCR are shown in Table 6. It can be seen that the target nucleic acid extract of Staphylococcus aureus is 1.1×10 6 Copies / μL.

[0193] [Table 4]

[0194] Reagents quantity <![CDATA[Platinum TM Taq DNA Polymerase]]> 0.125μL <![CDATA[10×PCR Buffer (-MgCl2)]]> 2.5μL <![CDATA[50mM MgCl2]]> 3μL dNTP mix (10 mM each) 2.5μL 10× Primer / Probe Mix 2.5μL template Specified amount water Make up the total volume to 25 μL total 25μL

[0195] [Table 5]

[0196]

[0197] [Table 6]

[0198]

[0199] [Standard DNA]

[0200] Standard DNA refers to a synthetic DNA fragment containing a nucleic acid amplification region of a primer for bacteria or a primer for fungi used this time. The outline method of the method for preparing the standard DNA used in this experiment is as follows. First, for bacteria, a portion of the sequence of Staphylococcus aureus (sequence number 59) and for fungi, a portion of the sequence of Candida albicans (sequence number 60) are introduced into a vector, respectively, and gene cloning is performed to obtain a standard DNA solution in which the target linear DNA fragment is dissolved by a restriction enzyme. Next, the concentration of the standard DNA solution is measured using an ultra-micro spectrophotometer NanoDrop (Thermo Fisher Scientific). Furthermore, the molecular weight of the standard DNA is used to calculate the number of copies of the standard DNA contained in the standard DNA solution (the copy concentration of the standard DNA solution). Specifically, use Figure 1 The calculation formula shown in .

[0201] [Test method]

[0202] The reaction solution used in the real-time PCR method was prepared as shown in Table 7. In addition, the oligonucleotide set shown in Table 1 was used, which included the forward primer, probe, and reverse primer used in the 10× primer / probe mixture (all manufactured by NIHON TECHNO SERVICE CO., LTD.). In addition, FAM was added to the 5' end of the probe, and BHQ1 was added to the 3' end. Real-time PCR was performed using CFX Connect TM The reaction conditions of the real-time PCR analysis system (Bio-Rad Laboratories) were heating at 95°C for 10 seconds, followed by 45 cycles of heating at 95°C for 10 seconds and at 66°C for 60 seconds (Table 8).

[0203] [Table 7]

[0204]

[0205] [Table 8]

[0206]

[0207] [Method for determining whether nucleic acid amplification reaction is inhibited]

[0208] First, when real-time PCR is performed on three or more samples with equal amounts of target nucleic acid, the amplification curves of these samples are considered to be arranged at substantially equal intervals. For example, when real-time PCR is performed using samples prepared at 10 times the target nucleic acid concentration in the form of 10 copies, 100 copies, and 1000 copies, the amplification curves of these samples are arranged at substantially equal intervals from the right. This means that a standard curve of the Cq value and the copy number using this real-time PCR can be prepared.

[0209] Furthermore, when the nucleic acid amplification reaction of the target nucleic acid is hindered, it can be judged by the amplification curve of the nucleic acid amplification reaction moving to the right. This is because when the nucleic acid amplification reaction of the target nucleic acid is hindered, the Cq value of the nucleic acid amplification reaction becomes larger. Therefore, compared with the amplification curve of the sample without adding human nucleic acid, the amplification curve of the sample with human nucleic acid added moves to the right, and it can be judged that the nucleic acid amplification reaction of the target nucleic acid is hindered by human nucleic acid. For example, when the amplification curve of the real-time PCR of the sample containing 1000 copies of the target nucleic acid of human nucleic acid is located near the amplification curve of the real-time PCR of the sample not containing 100 copies of the target nucleic acid of human nucleic acid, the detection sensitivity is reduced by about 1 / 10. In addition, for example, when the amplification curve of the real-time PCR of the sample containing 1000 copies of the target nucleic acid of human nucleic acid is located near the amplification curve of the real-time PCR of the sample not containing 10 copies of the target nucleic acid of human nucleic acid, the detection sensitivity is reduced to about 1 / 100 due to the addition of human nucleic acid. The hindering effect of the nucleic acid amplification reaction is judged according to the standards shown in Table 9.

[0210] [Table 9]

[0211]

[0212] [result]

[0213] The comparison of primers and probes used in each oligonucleotide set is shown in Table 10. The amplification curves using each oligonucleotide set are shown in Figure 2 to Figure 63 The inhibitory effect on nucleic acid amplification reaction is shown in Table 11, and whether a rapid sterility test can be performed in the presence of human nucleic acid is shown in Table 12. In addition, the base sequences of the probes and reverse primers of oligonucleotide sets 1 to 20 are extracted in Table 13, and the base sequences of the forward primers and reverse primers of oligonucleotide sets 21 to 36 are extracted in Table 14.

[0214] In addition, Figure 2 to Figure 63In the table, "●" represents the amplification curve of the real-time PCR of the sample without adding human nucleic acid, and "×" represents the amplification curve of the real-time PCR of the sample with added human nucleic acid, and each amplification curve is 1000 copies, 100 copies, and 10 copies from the left. Further, in Table 11, the situation where the target nucleic acid can be detected is evaluated as "0", and the situation where it cannot be detected is evaluated as "×", and "influence" represents the inhibitory effect of the nucleic acid amplification reaction. In addition, further, in Table 12, the situation where the presence of the target nucleic acid can be detected by a rapid sterility test in the presence of human nucleic acid is evaluated as "0", and the situation where the presence of the target nucleic acid cannot be detected by the rapid sterility test is evaluated as "×".

[0215] [Table 10]

[0216]

[0217] [Table 11]

[0218]

[0219] [Table 12]

[0220]

[0221] [Table 13]

[0222]

[0223] [Table 14]

[0224]

[0225] (Result of bacteria)

[0226] When no human-derived nucleic acid was added, 10 copies of the target nucleic acid were detected for oligonucleotide sets 1 to 20. The results when human-derived nucleic acid was added are shown below.

[0227] i) Whether sterility test can be performed (Table 12)

[0228] For oligonucleotide sets 1 to 20, 100 copies of the target nucleic acid were detected when 1000 ng of human nucleic acid was added. Furthermore, for oligonucleotide sets 1 to 2 and oligonucleotide sets 4 to 20, 10 copies of the target nucleic acid were detected when 1000 ng of human nucleic acid was added. For oligonucleotide set 3, although 10 copies of the target nucleic acid were not detected when 1000 ng of human nucleic acid was added, 10 copies of the target nucleic acid were detected when 100 ng of human nucleic acid was added.

[0229] From the above, it can be seen that even in the presence of human nucleic acid, oligonucleotide sets 1 to 20 can still be used for rapid sterility testing.

[0230] ii) Effect of inhibiting nucleic acid amplification reaction (Table 11)

[0231] For oligonucleotide set 1, oligonucleotide sets 4 to 12, and oligonucleotide sets 14 to 20, even when human nucleic acid was added, the detection sensitivity of the nucleic acid amplification reaction was not affected. For oligonucleotide set 2, oligonucleotide set 3, and oligonucleotide set 13, when human nucleic acid was added, the detection sensitivity of the nucleic acid amplification reaction decreased by about 10 times.

[0232] (Result of fungus)

[0233] For oligonucleotide sets 21 to 31 and oligonucleotide sets 34 to 36, when human nucleic acid was not added, 10 copies of the target nucleic acid were detected. For oligonucleotide set 32, when human nucleic acid was not added, 10 copies of the target nucleic acid were not detected, but 1000 copies of the target nucleic acid were detected. For oligonucleotide set 33, when human nucleic acid was not added, 10 copies of the target nucleic acid were not detected, but 100 copies of the target nucleic acid were detected. The results when human nucleic acid was added are shown below.

[0234] i) Whether sterility test can be performed (Table 12)

[0235] For oligonucleotide sets 21 to 24, oligonucleotide set 26, oligonucleotide set 27, oligonucleotide sets 29 to 31, and oligonucleotide sets 34 to 36, when 1000 ng of human nucleic acid was added, 10 copies of the target nucleic acid were detected. For oligonucleotide set 25, when 1000 ng of human nucleic acid was added, 1000 copies of the target nucleic acid were detected, although 100 copies of the target nucleic acid were not detected. In addition, for oligonucleotide set 25, when 100 ng of human nucleic acid was added, 10 copies of the target nucleic acid were detected. For oligonucleotide set 28, when 1000 ng of human nucleic acid was added, 10 copies of the target nucleic acid were not detected, but when 100 ng of human nucleic acid was added, 10 copies of the target nucleic acid were detected. For oligonucleotide set 33, when 1000 ng of human nucleic acid was added, 1000 copies of the target nucleic acid were not detected. In addition, for oligonucleotide set 33, when 100 ng of human nucleic acid was added, although 10 copies of the target nucleic acid could not be detected, 100 copies of the target nucleic acid were detected. For oligonucleotide set 32, when 1000 ng of human nucleic acid was added, 1000 copies of the target nucleic acid could not be detected. In addition, for oligonucleotide set 32, when 100 ng of human nucleic acid was added, although 100 copies of the target nucleic acid could not be detected, 1000 copies of the target nucleic acid were detected. Further, for oligonucleotide set 32, when 10 ng of human nucleic acid was added, although 10 copies of the target nucleic acid could not be detected, 1000 copies of the target nucleic acid were detected.

[0236] From the above, it can be seen that even in the presence of human nucleic acid, oligonucleotide sets 21 to 36 can still be used for rapid sterility testing.

[0237] ii) Effect of inhibiting nucleic acid amplification reaction (Table 11)

[0238] For oligonucleotide sets 21 to 24, oligonucleotide set 26, oligonucleotide sets 29 to 31, and oligonucleotide sets 35 to 36, even when human nucleic acids were added, there was no effect on the detection sensitivity of the nucleic acid amplification reaction. For oligonucleotide sets 27 and 34, when human nucleic acids were added, the detection sensitivity of the nucleic acid amplification reaction decreased by about 10 times. For oligonucleotide set 25, when human nucleic acids were added, the detection sensitivity of the nucleic acid amplification reaction decreased by about 100 times. For oligonucleotide sets 28, oligonucleotide sets 32, and oligonucleotide sets 33, when human nucleic acids were added, the detection sensitivity of the nucleic acid amplification reaction decreased by about 1,000 times or more.

[0239] Example 2

[0240] [Preparation of test samples]

[0241] The target microorganism was added to 1×10 cells derived from humans. 6 1 mL of the test sample was prepared by adjusting the concentration of Jurkat cells derived from adult leukemia T cells at 10 CFU / mL and 100 CFU / mL. The strains shown in Table 15 were used as target microorganisms.

[0242] [Pretreatment of test samples]

[0243] Next, free nucleic acids present outside the cells were removed by enzyme treatment based on Turbo Nuclease (endonuclease), and after inactivation of the enzyme, the cells were disrupted using EZ beads (AMR Inc.) to obtain a cell disrupted solution. Then, nucleic acids were extracted from the cell disrupted solution using QIAamp UCP DNA Micro Kit (QIAGEN KK) to obtain 40 μL of nucleic acid extract.

[0244] [Test method]

[0245] As shown in Table 16, a reaction solution used in the real-time PCR method was prepared. In addition, Table 15 shows the combination of the forward primer, probe, and reverse primer (all NIHONTECHNO SERVICE CO., LTD.) used in the 10× primer / probe mixture. In addition, for the probe, FAM was added to the 5' end and BHQ 1 was added to the 3' end. Real-time PCR used CFXConnect (Bio-Rad Laboratories), and the reaction conditions were heating at 95°C for 10 seconds, followed by 45 cycles of 95°C for 10 seconds and 66°C for 60 seconds (Table 17). In addition, the test was performed twice.

[0246] [Table 15]

[0247]

[0248] [Table 16]

[0249] Reagents quantity <![CDATA[Platinum TM Taq DNA Polymerase, DNA-free]]> 0.25μL <![CDATA[10×PCR Buffer (-MgCl2)]]> 2.5μL <![CDATA[50mM MgCl2]]> 0.75μL dNTP mix (10 mM each) 0.5μL 10× Primer / Probe Mix 2.5μL Nucleic acid extraction solution 10μL water 8.5μL total 25μL

[0250] [Table 17]

[0251]

[0252] [result]

[0253] The amplification curve using bacteria is shown in Figures 64 to 71 , the amplification curve using fungi is shown in Figure 72 to Figure 75 Table 18 shows whether sterility tests using bacteria and fungi can be performed.

[0254] In addition, in this test, 10 μL of the 40 μL nucleic acid extract was used for real-time PCR, so the amount of human-derived cells and target microorganisms contained in the test sample was 1 / 4 of the amount in the test sample. In addition, in Table 18, the situation where the presence of the target microorganism can be detected by the rapid sterility test in the presence of human-derived cells is evaluated as "0", and the situation where the presence of the target microorganism cannot be detected by the rapid sterility test is evaluated as "×". The results show that in the test 2 of the two tests, the presence of the target microorganism can be detected as "2 / 2", and in the test 1 of the two tests, the presence of the target microorganism can be detected as "1 / 2".

[0255] [Table 18]

[0256]

[0257] Can the sterility test of bacteria be performed (Table 18)

[0258] For oligonucleotide set 20, in test 2 of two tests, 25 CFU of the target microorganism contained in the test sample to which human-derived cells were added could be detected. Furthermore, for oligonucleotide set 20, in test 1 of two tests, 2.5 CFU of the target microorganism contained in the test sample to which human-derived cells were added could be detected. However, it is believed that the reason for failure to detect was that the amount of the target microorganism contained in the PCR was a very small amount of 2.5 CFU / test.

[0259] As described above, it can be seen that the oligonucleotide set 20 can be used for a rapid sterility test even when a test sample to which human-derived cells are added is used.

[0260] Capable of performing fungal sterility tests (Table 18)

[0261] Oligonucleotide set 21 was able to detect 25 CFU and 2.5 CFU of the target microorganism contained in the test sample to which human-derived cells were added in test 2 of the two tests. As can be seen from the above, even when a test sample to which human-derived cells were added was used, oligonucleotide set 21 can still be used for a rapid sterility test.

[0262] Industrial Applicability

[0263] By using the present invention, it is possible to reduce the hindrance of the nucleic acid amplification reaction of the target nucleic acid by the human nucleic acid in the nucleic acid amplification method, to detect bacterial or fungal nucleic acids in a comprehensive manner, and to use it for rapid sterility tests in regenerative medicine.

Claims

1. A rapid sterility test method for detecting the presence of bacterial nucleic acid contained in a test sample containing human nucleic acid by a nucleic acid amplification method, characterized in that: The oligonucleotide set used in this nucleic acid amplification method is: (1) one or more probes selected from probe group PM1, probe group PM2 and probe group PM3, wherein the probe group PM1 comprises the base sequence represented by sequence number 18 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in the probe group PM1 is 12 to 42, the probe group PM2 comprises the base sequence represented by sequence number 27 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in the probe group PM2 is 8 to 42, and the probe group PM3 comprises the base sequence represented by sequence number 30 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in the probe group PM3 is 7 to 42; and (2) One or more reverse primers in a primer set RM, wherein the primer set RM comprises the base sequence represented by SEQ ID NO: 4 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the primer set RM is 8 to 30.

2. The rapid sterility test method according to claim 1, characterized in that: The probe is one or more probes selected from the base sequences represented by SEQ ID NOs: 21 to 26, SEQ ID NO: 29 or SEQ ID NO: 32, or base sequences obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences, The reverse primer is one or more reverse primers selected from the base sequences represented by SEQ ID NOs: 7 to 17, or base sequences in which 1 to 3 bases are inserted, deleted or substituted in each of these base sequences.

3. The rapid sterility test method according to claim 1 or 2, characterized in that: The oligonucleotide set further comprises: (3) One or more forward primers in primer set FM, wherein the primer set FM comprises the base sequence represented by SEQ ID NO: 1 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the primer set FM is 13 to 50.

4. The rapid sterility test method according to claim 3, characterized in that: The forward primer is one or more forward primers selected from the base sequence represented by SEQ ID NO: 3 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence.

5. The rapid sterility test method according to any one of claims 1 to 4, characterized in that: The human nucleic acid contained in the sample is 1 to 1×10 5 ng.

6. The rapid sterility test method according to any one of claims 1 to 5, characterized in that: The number of copies of the detected bacterial nucleic acid ranges from 1 to 100 copies.

7. The rapid sterility test method according to any one of claims 1 to 6, characterized in that: The rapid sterility test method uses a nucleic acid extract obtained by adding a nucleic acid degrading enzyme to a test sample that contains at least cells from humans as human nucleic acid and bacteria as bacterial nucleic acid, and extracting nucleic acids contained in human cells and / or bacteria from an enzyme-treated sample from which the nucleic acid degrading enzyme has been removed and / or inactivated.

8. The rapid sterility test method according to claim 7, characterized in that: The number of bacteria detected was 1 to 100 CFU.

9. A rapid sterility test method for detecting the presence of fungal nucleic acid contained in a test sample containing human nucleic acid by a nucleic acid amplification method, characterized in that: The oligonucleotide set used in this nucleic acid amplification method is: (1) one or more forward primers in primer set FY1 and primer set FY2, wherein the primer set FY1 comprises the base sequence represented by SEQ ID NO: 33 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in primer set FY1 is 11 to 44, and the primer set FY2 comprises the base sequence represented by SEQ ID NO: 45 or a base sequence obtained by inserting, deleting or replacing 1 to 3 bases in the base sequence, and the number of bases in primer set FY2 is 16 to 44; and (2) One or more reverse primers in a primer set RY, wherein the primer set RY comprises the base sequence represented by SEQ ID NO: 51 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the primer set RY is 17 to 44.

10. The rapid sterility test method according to claim 9, characterized in that: The forward primer is one or more forward primers selected from the base sequences represented by SEQ ID NOs: 37 to 44, or 46 to 48, or base sequences obtained by inserting, deleting or replacing 1 to 3 bases in each of these base sequences, The reverse primer is one or more reverse primers selected from the base sequences represented by SEQ ID NOs: 52 to 55, or base sequences in which 1 to 3 bases are inserted, deleted or substituted in each of these base sequences.

11. The rapid sterility test method according to claim 9 or 10, characterized in that: The oligonucleotide set further comprises: (3) One or more probes in a probe set PY, wherein the probe set PY comprises the base sequence represented by SEQ ID NO: 56 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence, and the number of bases in the probe set PY is 17 to 50.

12. The rapid sterility test method according to claim 11, characterized in that: The probe is one or more probes selected from the base sequence represented by SEQ ID NO: 58 or a base sequence in which 1 to 3 bases are inserted, deleted or substituted in the base sequence.

13. The rapid sterility test method according to any one of claims 9 to 12, characterized in that: The human nucleic acid contained in the sample is 1 to 1×10 5 ng.

14. The rapid sterility test method according to any one of claims 9 to 13, characterized in that: The copy number of the detected fungal nucleic acid ranged from 1 to 100 copies.

15. The rapid sterility test method according to any one of claims 9 to 14, characterized in that: The rapid sterility test method uses a nucleic acid extract obtained by adding a nucleic acid degrading enzyme to a test sample that contains at least cells from humans as human nucleic acid and fungi as fungal nucleic acid, and extracting nucleic acids contained in human cells and / or fungi from an enzyme-treated sample from which the nucleic acid degrading enzyme has been removed and / or inactivated.

16. The rapid sterility test method according to claim 15, characterized in that: The number of fungi detected was 1 to 100 CFU.

17. The rapid sterility test method according to any one of claims 7, 8, 15 and 16, characterized in that: Cells from humans are 1 to 1×10 7 cells.

18. The rapid sterility test method according to any one of claims 1 to 17, characterized in that: The nucleic acid amplification method is PCR or real-time PCR.

19. A real-time PCR kit, wherein: The kit is used for the rapid sterility test method according to any one of claims 1 to 18.

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