Method for sampling arbitrary base or peptide sequence

Abstract

When detecting a target that is present in only a trace amount in a sample, it is necessary to at the same time verify that highly sensitive detection is being performed with high accuracy; however, there has been no development of a method capable of verifying whether highly sensitive detection is being performed with high accuracy.　The inventors of the present invention have solved the above problem by demonstrating that when detecting a target that is present in only a trace amount in a sample, by verifying the detection in a control sample including a positive control under the same conditions as the subject sample, it is possible to demonstrate that the detection process is functioning properly, and as a result, it is possible to confirm that highly sensitive detection of the target that is present in only a trace amount in the sample is being achieved.

Description

A method for sampling any base or peptide sequence

[0001] The present invention relates to providing a method for detecting trace amounts of nucleic acid molecules or peptide molecules in a test sample in vitro and simultaneously confirming the accuracy of the reaction.

[0002] In recent years, new methods have been developed or improved for the highly sensitive detection of targets present in a sample at the nucleic acid or protein level.

[0003] For example, a highly sensitive technique for visualizing the localization of bacteria, which are known to be present only in small amounts within tumor tissue, is the in situ hybridization method (RNA scope hybridization) for RNA sequences specific to bacteria (species). TM ) has been developed (Wang F, et al., J Mol Diagn. 2012, PMID: 22166544) and is commercially available as a laboratory reagent.

[0004] At the protein level, immunostaining methods are used to detect target antigens present in trace amounts in a sample or bacteria or cells containing the target antigen with high sensitivity using antibodies against the target antigen.

[0005] Both methods are highly useful because they can detect targets present in a sample with high sensitivity, but they have the drawback of not being able to confirm that the target was not present in the sample.

[0006] Thus, although highly sensitive detection is now possible, a method that can simultaneously verify accuracy has not yet been developed, and there is a need to establish a method that can reliably detect targets that are present in only trace amounts in a sample.

[0007] Wang F, et al., J Mol Diagn. 2012, PMID: 22166544

[0008] When detecting a target that is present in very small amounts in a sample, it is necessary to simultaneously verify that highly sensitive detection is being carried out with high accuracy, but no method has been developed that can verify whether highly sensitive detection is being carried out with high accuracy.

[0009] The inventors of the present invention have demonstrated that when detecting a target that is present in very small amounts in a sample, it is possible to demonstrate that the detection process is functioning correctly by detecting a control sample containing a positive control under the same conditions as the test sample, and as a result, it is possible to confirm that a target that is present in very small amounts in a sample can be detected with high sensitivity, thereby solving the above-mentioned problem.

[0010] More specifically, to solve the above-mentioned problems, the present application provides the following aspects: [1]: A positive control sample containing a non-pathogenic nucleic acid molecule containing a target base sequence detected by an ISH probe used in a nucleic acid in situ hybridization (ISH) method, or a non-pathogenic peptide molecule or non-pathogenic protein molecule containing a target amino acid sequence detected by immunostaining. [2]: The sample according to [1], in which a non-pathogenic nucleic acid molecule containing a target sequence detected by an ISH probe is introduced into a competent cell via a vector. [3]: The sample according to [1] or [2], in a nucleic acid in situ hybridization (ISH) method, the sample is used to confirm the reaction of the detection system of the ISH method performed on the test sample by performing the ISH method on the positive control sample under the same conditions as the test sample. [4]: The sample according to [1], wherein a non-pathogenic peptide molecule or non-pathogenic protein molecule containing a target sequence to be detected by immunostaining is a nucleic acid molecule encoding the peptide molecule or non-pathogenic protein molecule introduced into a competent cell via a vector and expressed. [5]: The sample according to [1] or [4], for confirming the reaction of the detection system of the immunostaining method performed on a test sample by performing immunostaining on a positive control sample under the same conditions as for the test sample in an immunostaining method for peptide molecules or protein molecules. [6]: A method of using a control sample (B) in a nucleic acid in situ hybridization (ISH) method, for confirming the reaction of the detection system under the same conditions as for the test sample by performing ISH on (A) the test sample and (B) a control sample containing a non-pathogenic nucleic acid molecule containing a target base sequence to be detected by an ISH probe under the same conditions. [7]: The method according to [6], in which the ISH method for nucleic acids is an ISH method for RNA (RNA ISH method) or an ISH method for DNA (DNA ISH method). [8]: The method according to [6] or [7], in which the reaction confirmation of the detection system is to confirm that it is not affected by imperfections in the experimental technique.[9]: The method according to [6] or [7], wherein the non-pathogenic nucleic acid molecule containing the target base sequence in the (B) control sample is obtained by artificial synthesis, viral synthesis, bacterial synthesis, or cellular synthesis.

[10] : A method for using a (B) control sample in an immunostaining method for peptide molecules or proteins, to confirm the reaction of the detection system under the same conditions as those for evaluating the (A) test sample, by performing an immunostaining method under the same conditions on (A) the test sample, and (B) a control sample expressing a non-pathogenic peptide molecule or non-pathogenic protein containing the target amino acid sequence detected by the antibody or antibody derivative used.

[11] : The method according to

[10] , wherein the reaction confirmation of the detection system is confirmed to be free from the influence of imperfections in the experimental technique.

[12] : The method according to

[10] or

[11] , wherein the non-pathogenic peptide molecule or non-pathogenic protein molecule containing the target amino acid sequence in the (B) control sample is obtained by artificial synthesis, viral synthesis, bacterial synthesis, or cellular synthesis.

[0011] When detecting a target that is present in very small amounts in a sample, the present invention can demonstrate that the detection process is functioning correctly by detecting a control sample containing a positive control under the same conditions as the test sample, thereby providing a new method that can confirm that a target that is present in very small amounts in a sample can be detected with high sensitivity.

[0012] Figure 1 shows the nucleotide sequence specific to Fusobacterium nucleatum (F. nucleatum) (gttgcaatcg aacgtaagag tctaggattt ccgaatggag caatctatta agatggagtc ttaatacgaa agagggaacc acgtgaactg aaacatctaa gtaacgtgag gaaaagaaag taaaaacgat acccaaagta gcggcgagcg aactgggtca agcctaaacc ttaaatatgt caaggataca gccgttgtat ttaaggggtt gagggacaaa gtagtgaaga actgtaagat attcaatata gtgtattgat gaattagaat tgtatggaaa gataaaccgc agaaggtgag agtcctgtat aagtaaatct ttacacatat aactttgctc ccaag, SEQ ID NO: 1, Accession No. CP003723.1). Figure 1A shows the results of RNA ISH on E. coli-embedded sections of a control sample, while Figure 1B shows the results of RNA ISH on E. coli-embedded sections of a negative control sample. Figure 1C shows the results of RNA ISH on liver tumor tissue sections to visualize the presence or absence of F. nucleatum. Figure 2 shows the results of RNA ISH using a Bacillus-specific sequence (ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga aaccggagct aataccggat agttccttga accgcatggt tcaaggatga aagacggttt cggctgtcac ttacagatgg acccgcggcg cattagctag ttggtggggt aatggctcac caaggcgacg atgcgtagcc gacctgagag g, SEQ ID NO: 2, Accession No. AB109633.1) as the target sequence. Figure 2A shows the results of RNA ISH performed on E. coli-embedded sections of a control sample, and Figure 2B shows the results of RNA ISH performed on E. coli-embedded sections of a negative control sample.

[0013] As mentioned above, in recent years, there has been a need to detect targets that are present in very small amounts in a sample with high sensitivity at the nucleic acid or protein level, and to confirm that the detection is carried out with high accuracy.

[0014] In order to solve such problems, the present invention provides a positive control sample that can be used to detect targets that are present in very small amounts in a sample, and by detecting a control sample containing this positive control sample under the same conditions as the test sample, it can be shown that the detection process is functioning correctly, and as a result, it has been confirmed that targets that are present in very small amounts in a sample can be detected with high sensitivity.

[0015] In a first aspect, the present invention can provide a positive control sample comprising a non-pathogenic nucleic acid molecule containing a target base sequence to be detected by an ISH probe used in a nucleic acid in situ hybridization (ISH) method, or a non-pathogenic peptide molecule or non-pathogenic protein molecule containing a target amino acid sequence to be detected by immunostaining.

[0016] When the positive control sample of the present invention contains a non-pathogenic nucleic acid molecule containing a target base sequence detected by an ISH probe used in a nucleic acid in situ hybridization (ISH) method, this means that the nucleic acid molecule itself or a peptide or protein expressed based on this nucleic acid molecule is non-pathogenic. Even when the target of detection in the nucleic acid ISH method is a pathogenic microorganism (virus or bacteria), the positive control can be non-pathogenic, thereby eliminating safety and management risks when performing the nucleic acid ISH method.

[0017] When the positive control sample of the present invention contains a non-pathogenic peptide molecule or non-pathogenic protein molecule containing the target amino acid sequence to be detected by immunostaining, this means that the peptide molecule or protein molecule itself is non-pathogenic. Even if the target to be detected by immunostaining is a pathogenic microorganism (virus or bacteria), the positive control can be non-pathogenic, thereby eliminating safety and management risks when performing immunostaining.

[0018] Here, pathogenicity refers to the property or ability to cause harmful events such as infectious diseases in humans. For example, in the case of a pathogenic microorganism, it means that a laboratory management system of P1 level or higher is required.

[0019] In the ISH method for nucleic acids, non-pathogenic nucleic acid molecules containing the target sequence detected by the ISH probe can be introduced into competent cells via a vector. Here, "competent cells" refers to cells that are capable of incorporating foreign DNA (e.g., plasmid or phage DNA). Methods commonly used in the art can be used to introduce a vector into competent cells. In the present invention, a vector incorporating an isolated nucleic acid containing the target sequence detected by the ISH probe is introduced into competent cells, and the competent cells containing this vector are then grown to amplify the non-pathogenic nucleic acid molecule containing the target sequence detected by the ISH probe. The amplified non-pathogenic nucleic acid molecule can be detected with the ISH probe to serve as a positive control.

[0020] As a use of the non-pathogenic nucleic acid molecule of the present invention, in the ISH method for nucleic acids, the ISH method can be performed on a positive control sample under the same conditions as the test sample, thereby confirming the reaction of the detection system of the ISH method performed on the test sample.

[0021] In conventional methods in the technical field, when a target sequence is not detected from a test sample when performing the ISH method on a nucleic acid, it is difficult to determine whether the target sequence is not detected because the target sequence of interest is not present in the test sample, or whether the reaction of the detection system of the ISH method performed on the test sample is not performed properly.

[0022] In the present invention, when the ISH method for nucleic acids is performed on a positive control sample under the same conditions as for the test sample, if the target sequence cannot be detected from the positive control sample, it indicates that the reaction of the detection system for the ISH method performed was not performed properly, and if the target sequence can be detected from the positive control sample, it indicates that the reaction of the detection system for the ISH method performed was performed properly.

[0023] Furthermore, by calculating the theoretical value of the concentration or copy number of the nucleic acid molecule containing the target base sequence to be contained in the positive control sample, and then performing the ISH method on the positive control sample to measure the actual value, it is possible to confirm whether the theoretical value and the actual value are consistent, and also to confirm the quantitativeness of the reaction of the detection system of the ISH method performed.

[0024] A non-pathogenic peptide molecule or non-pathogenic protein molecule containing a target sequence to be detected by immunostaining can be obtained by introducing a nucleic acid molecule encoding the peptide molecule or non-pathogenic protein molecule into competent cells using a vector and expressing the molecule. Here, "competent cells" refers to cells that are capable of incorporating foreign DNA (such as plasmid or phage DNA) into the cells. Methods commonly used in the art can be used for introducing and expressing the molecule into competent cells using a vector. Specifically, in the present invention, a vector incorporating an isolated nucleic acid encoding a non-pathogenic peptide molecule or non-pathogenic protein molecule containing a target sequence to be detected by immunostaining can be introduced into competent cells, and the peptide molecule or protein can be expressed by the competent cells transfected with this vector. The expressed peptide molecule or protein can then be detected with an antibody, providing a positive control.

[0025] A sample according to [1] or [4], for confirming the reaction of the detection system of the immunostaining method performed on the test sample (A) by performing the immunostaining method on the test sample (A) under the same conditions as the test sample in an immunostaining method for peptide molecules or protein molecules.

[0026] As an application of the non-pathogenic peptide molecule or protein molecule of the present invention, in an immunostaining method, by performing immunostaining on a positive control sample under the same conditions as the test sample, the reaction of the detection system of the immunostaining method performed on the test sample can be confirmed.

[0027] In conventional methods in the technical field, when a target molecule (peptide molecule or protein molecule) is not detected in a test sample when immunostaining is performed, it is difficult to determine whether the target molecule is not detected because the target molecule of interest is not present in the test sample, or whether the reaction of the detection system in the immunostaining performed on the test sample is not performed appropriately.

[0028] In the present invention, when immunostaining is performed on a positive control sample under the same conditions as for the test sample, if the target molecule cannot be detected from the positive control sample, it indicates that the reaction of the detection system in the immunostaining method performed was not performed properly, and if the target molecule can be detected from the positive control sample, it indicates that the reaction of the detection system in the immunostaining method performed was performed properly.

[0029] Furthermore, by calculating the theoretical value of the concentration or number of target molecules contained in a positive control sample and then performing immunostaining on the positive control sample to measure the actual value, it is possible to confirm whether the theoretical value and the actual value are consistent, and also to confirm the quantitativeness of the reaction of the detection system of the performed immunostaining method.

[0030] In a second aspect, the present invention provides a method for using the positive control sample of the first aspect described above as a means for confirming the reaction of the detection system in the above-mentioned highly sensitive detection at the nucleic acid level, in a nucleic acid in situ hybridization method (ISH method), by performing the ISH method under the same conditions on (A) a test sample, and (B) a control sample containing a non-pathogenic nucleic acid molecule containing a target base sequence to be detected by the ISH probe, thereby using (B) a control sample to evaluate the test sample and confirm the reaction of the detection system under the same conditions.

[0031] In the art, the ISH method of nucleic acids can detect target nucleic acid molecules present in very small amounts in a test sample with high sensitivity. In the present invention, the ISH method of nucleic acids may refer to the ISH method for RNA (RNA ISH method) or the ISH method for DNA (DNA-ISH method). For example, the RNA ISH method for detecting RNA as a target nucleic acid molecule can be performed by a method commonly used in the art.

[0032] As mentioned above, in conventional methods in the technical field, when a target sequence is not detected from a test sample when performing the ISH method on a nucleic acid, it is difficult to determine whether the target sequence is not detected because the target sequence of interest is not present in the test sample, or whether the reaction of the detection system for the ISH method performed on the test sample is not performed properly.

[0033] In the present invention, when performing the ISH method on a nucleic acid in a test sample, the ISH method is performed on a control sample containing the target nucleic acid molecule to be detected by the ISH probe under the same conditions as the test sample to detect the target base sequence, thereby making it possible to confirm whether the reaction confirmation of the detection system in the ISH method of the test sample is affected by imperfections in the experimental technique.

[0034] The probe used in the ISH method for nucleic acids is a nucleic acid molecule having a base sequence that can bind (hybridize) with the target base sequence of the target nucleic acid molecule to be detected. In other words, the probe can complementarily bind (hybridize) with a base sequence that is present in the base sequence of the target nucleic acid molecule but not present in other nucleic acid molecules. Such a target base sequence can be a nucleic acid sequence that is specific to the nucleic acid of the target (e.g., virus, bacteria, cell) to be detected (present in that nucleic acid but not present in other targets).

[0035] In the present invention, the term "non-pathogenic nucleic acid molecule containing a target nucleotide sequence detected by an ISH probe" refers to a non-pathogenic nucleic acid molecule containing a nucleotide sequence that identifies the test sample to be detected by the ISH probe, in other words, a non-pathogenic nucleic acid molecule containing a nucleotide sequence that is specifically present in the target. More specifically, a non-pathogenic nucleic acid molecule containing a nucleotide sequence that is present in the target but not present in other nucleic acid molecules, or a non-pathogenic nucleic acid molecule containing such a nucleotide sequence and to which the probe can detectably bind. The nucleotide sequence specifically present in the target to be detected may be a nucleotide sequence already known in the art to be specific for the target to be detected, or a newly specific nucleotide sequence may be selected based on the nucleotide sequence of the target to be detected. Here, the sequence that can be detected by the probe may have nucleotide mutations (deletions, substitutions, or additions) in the target nucleotide sequence, for example, one or several nucleotide mutations in the target nucleotide sequence, as long as the probe can hybridize. Furthermore, when designing a probe, it is possible to anticipate mutations (deletions, substitutions, additions) in the bases of the target base sequence and design a probe that has mutations (deletions, substitutions, additions) in one or several bases in a sequence complementary to the target base sequence.

[0036] The "non-pathogenic nucleic acid molecule containing the target base sequence detected by the ISH probe" contained in such a control sample may be of any origin, such as an artificially synthesized nucleic acid molecule or a nucleic acid molecule expressed in a virus, bacterium, or cell using a vector construct, as long as it is non-pathogenic.

[0037] When a "non-pathogenic nucleic acid molecule containing a target base sequence detected by an ISH probe" is provided as an artificially synthesized nucleic acid molecule, it can be produced by designing the entire base sequence of the non-pathogenic nucleic acid molecule containing the target base sequence and synthesizing the sequence using a method used in the relevant technical field, such as a nucleotide synthesis method.

[0038] Furthermore, when providing a "non-pathogenic nucleic acid molecule containing a target base sequence to be detected by an ISH probe" as one expressed using a vector construct, the non-pathogenic nucleic acid molecule containing the target base sequence can be inserted into a vector capable of functioning in the virus, bacterium, or cell in which the sequence is desired to be expressed so that it can be expressed, and the "non-pathogenic nucleic acid molecule containing a target base sequence to be detected by an ISH probe" can be provided by expressing it in the virus, bacterium, or cell.

[0039] In the present invention, a non-pathogenic nucleic acid molecule containing a target nucleotide sequence can be included in a control sample and used as a positive control when performing ISH. Therefore, the theoretical concentration or copy number of the nucleic acid molecule containing the target nucleotide sequence to be included in the (B) control sample is calculated, and the (B) control sample is subjected to ISH under the same conditions as those used to perform the ISH on the (A) test sample to confirm whether the calculated concentration or copy number of the nucleic acid molecule containing the target nucleotide sequence in the (B) control sample can be detected. In this way, by confirming whether the theoretical value matches the actual measured value, it is possible to verify that the target nucleotide sequence contained in the (A) test sample has been detected qualitatively or quantitatively without being affected by imperfections in the experimental technique.

[0040] (B) If the ISH method is performed technically correctly using a control sample and the reaction of the detection system can be confirmed, the detection result of the (A) test sample performed under the same conditions is proven to be the result of the ISH method performed technically correctly.If a nucleic acid molecule containing the detected target base sequence is not present, the possibility that detection was not possible due to improper experimental technique is ruled out, and it can be proven that a nucleic acid molecule containing the target base sequence is not present in the (A) test sample.

[0041] The concept that by including a target substance in a (B) control sample and performing the detection process for the (B) control sample under the same conditions as the (A) test sample, it is possible to accurately prove the presence or absence of a target substance that may be contained in the (A) test sample can also be used in various detection methods other than the ISH method of nucleic acids.

[0042] In the present invention, a similar technique can also be applied to the detection of peptide molecules or proteins in a test sample as one of various detection methods in other embodiments. That is, in a third embodiment of the present invention, as a use of the positive control sample of the first embodiment described above, as a means for confirming the reaction of the detection system in the above-mentioned highly sensitive detection at the peptide molecule level or protein level, in an immunostaining method for peptide molecules or proteins, (A) the test sample, and (B) a control sample expressing a non-pathogenic peptide molecule or non-pathogenic protein containing the target amino acid sequence detected by the antibody or antibody derivative used, are subjected to the immunostaining method under the same conditions as those for evaluating the test sample (A), thereby using the control sample (B) to confirm the reaction of the detection system under the same conditions as those for evaluating the test sample (A).

[0043] In the art, target peptide molecules or target proteins present in very small amounts in a test sample can be detected with high sensitivity by immunostaining of peptide molecules or proteins, which can be performed by techniques commonly used in the art.

[0044] As described above, in conventional methods in the technical field, when a target molecule is not detected from a test sample when an immunostaining method is performed, it is difficult to determine whether the target molecule is not detected because the target molecule of interest is not present in the test sample, or whether the target molecule is not detected because the reaction of the detection system in the immunostaining method performed on the test sample is not performed appropriately.

[0045] In the present invention, when performing an immunostaining method for peptide molecules or proteins on a test sample, a control sample containing the target peptide molecules or protein molecules to be detected is subjected to immunostaining under the same conditions as the test sample to detect the target amino acid sequence, thereby making it possible to confirm whether the reaction confirmation of the detection system in the immunostaining method for the test sample is affected by imperfections in the experimental technique.

[0046] In immunostaining of peptide molecules or proteins, target peptide molecules or protein molecules can be detected using antibodies or antibody derivatives that specifically bind to the target peptide molecules or protein molecules. That is, the antibodies or antibody derivatives used to detect target peptide molecules or protein molecules can bind to amino acid sequences that are present in the amino acid sequence of the target peptide molecule or protein molecule but not present in other peptide molecules or protein molecules. Such target amino acid sequences can be amino acid sequences that are specific to peptide molecules or protein molecules contained in the target (e.g., virus, bacteria, cell) to be detected (i.e., present in the peptide molecule or protein molecule but not present in other targets).

[0047] In the present invention, the term "non-pathogenic peptide molecule or non-pathogenic protein containing a target amino acid sequence detected by an antibody or antibody derivative" refers to a non-pathogenic peptide molecule or non-pathogenic protein containing an amino acid sequence that identifies the test sample to be detected by the antibody or antibody derivative, in other words, a non-pathogenic peptide molecule or non-pathogenic protein containing an amino acid sequence that is specifically present in the target, more specifically, a non-pathogenic peptide molecule or non-pathogenic protein containing an amino acid sequence that is present in the target peptide molecule or protein but not in other peptide molecules or proteins, or a sequence containing that amino acid sequence that can be detectably bound by an antibody or antibody derivative. The amino acid sequence specifically present in the target to be detected may be an amino acid sequence already known in the art to be specific for the target to be detected, or a newly specific amino acid sequence may be selected based on the amino acid sequence of the target to be detected. Here, the sequence that can be detected by the antibody may have amino acid mutations (deletions, substitutions, additions) in the target amino acid sequence to the extent that the antibody can detectably bind, for example, the target amino acid sequence may have one or several amino acid mutations (deletions, substitutions, additions). Furthermore, when selecting an antibody, it is also possible to assume amino acid mutations (deletions, substitutions, additions) in the target amino acid sequence and select an antibody that can detectably bind to a target amino acid sequence that has mutations (deletions, substitutions, additions).

[0048] The "non-pathogenic peptide molecule or non-pathogenic protein containing the target amino acid sequence detected by the antibody or antibody derivative" contained in such a control sample may be of any origin, such as an artificially synthesized peptide molecule or protein, or a peptide molecule or protein expressed in a virus, bacterium, or cell using a vector construct containing a nucleic acid molecule encoding the peptide molecule or protein, as long as it is non-pathogenic.

[0049] When a "non-pathogenic peptide molecule or non-pathogenic protein containing a target amino acid sequence detected by an antibody or antibody derivative" is provided as an artificially synthesized amino acid molecule, it can be produced by designing the entire amino acid sequence of the non-pathogenic peptide molecule or non-pathogenic protein containing the target amino acid sequence and synthesizing the sequence using a method used in the art, such as solid-phase peptide synthesis.

[0050] Furthermore, when providing a "non-pathogenic peptide molecule or non-pathogenic protein comprising a target amino acid sequence detected by an antibody or antibody derivative" as one expressed using a vector construct, the nucleic acid molecule encoding the non-pathogenic peptide molecule or non-pathogenic protein can be inserted into a vector capable of functioning in the virus, bacterium, or cell in which the sequence is desired to be expressed so that the "non-pathogenic peptide molecule or non-pathogenic protein comprising the target amino acid sequence" can be expressed in the virus, bacterium, or cell.

[0051] In the present invention, non-pathogenic peptide molecules or non-pathogenic proteins containing the target amino acid sequence can be included in a control sample and used as a positive control when performing immunostaining. Therefore, the theoretical concentration or number of molecules of the peptide molecule or protein containing the target amino acid sequence to be included in the (B) control sample is calculated, and the (B) control sample is subjected to immunostaining under the same conditions as those used to perform immunostaining on the (A) test sample to confirm whether the calculated concentration or number of molecules of the peptide molecule or protein containing the target amino acid sequence in the (B) control sample can be detected. In this way, by confirming whether the theoretical value and the actual measured value are consistent, it is possible to verify that the target amino acid sequence contained in the (A) test sample has been detected qualitatively or quantitatively, without being affected by imperfections in the experimental technique.

[0052] (B) If the immunostaining method is performed technically correctly using a control sample and the reaction of the detection system can be confirmed, the detection result of the (A) test sample performed under the same conditions is proven to be the result of the immunostaining method performed technically correctly.If there are no peptide molecules or proteins containing the target amino acid sequence to be detected, the possibility that the detection was not possible due to improper experimental techniques is eliminated, and it can be proven that there are no peptide molecules or proteins containing the target base sequence in the (A) test sample.

[0053] The present invention will be specifically illustrated by the following examples, which are not intended to limit the present invention in any way.

[0054] Example 1: Verification of RNA ISH method using target base sequence (1) In this example, experiments were conducted using F. nucleatum as a model detection target to verify the detection accuracy of the ISH method for detecting its localization in tumor tissue.

[0055] For the RNA ISH method, we used RNA scope PCR targeting the F. nucleatum specific base sequence (SEQ ID NO: 1, Accession No. CP003723.1) to detect F. nucleatum potentially present in tumors. TM The presence of the above-mentioned base sequence specific to F. nucleatum was detected using the method (Wang F, et al., J Mol Diagn. 2012, PMID: 22166544).

[0056] To investigate the intratumoral localization of F. nucleatum, we performed RNA ISH using the aforementioned ISH probe targeting an F. nucleatum -specific sequence (SEQ ID NO: 1, Accession No. CP003723.1) to visualize the presence or absence of F. nucleatum in liver tumor tissue sections (Fig. 1C).

[0057] When performing RNA ISH on the test sample, we also performed RNA ISH on a control sample under the same conditions as the test sample. The control sample was prepared by introducing a base sequence specific to F. nucleatum (SEQ ID NO: 1, Accession No. CP003723.1) into E. coli.

[0058] Specifically, a F. nucleatum-specific sequence (SEQ ID NO: 1, Accession No. CP003723.1) was inserted into the pUCFa vector to generate a vector. This vector was then inserted into E. coli and the cells were cultured. After the culture, the cells were centrifuged at 6,000 × g for 10 minutes, fixed in formalin, embedded in paraffin (15 × 15 × 5 mm), and thinly sliced ​​to prepare tissue sections suitable for use as control samples in RNA ISH. RNA ISH was also performed on these control samples under the same conditions as the test samples. As a negative control, E. coli cells not transfected with the F. nucleatum-specific sequence (SEQ ID NO: 1, Accession No. CP003723.1) were subjected to RNA ISH under the same conditions as the test samples.

[0059] The results are shown in Figure 1. Figure 1A shows the results of RNA ISH using a sequence specific to F. nucleatum as the target sequence. Specifically, RNA ISH performed on the control sample revealed a positive image (arrow) of the F. nucleatum-specific sequence (Figure 1A). No positive image was detected in the negative control sample. This indicates that the F. nucleatum-specific sequence (SEQ ID NO: 1, Accession No. CP003723.1) expressed in E. coli as the control sample was detected as a positive image by the ISH probe, demonstrating that the RNA ISH performed was performed accurately and without any experimental errors.

[0060] On the other hand, when RNA ISH was performed on the test sample of tumor tissue, a positive image (arrow) for F. nucleatum was also observed in the test sample by RNA ISH (Fig. 1B).

[0061] The results of the RNA ISH method on the control sample confirmed that the experimental technique was flawless and that the RNA ISH method was performed accurately. Therefore, when the RNA ISH method was performed on the test sample under the same conditions, a base sequence specific to F. nucleatum (SEQ ID NO: 1, Accession No. CP003723.1) was detected in the test sample (Figure 1C), indicating the presence of F. nucleatum in the test sample.

[0062] Example 2: Verification of RNA ISH method using target base sequence (2) In this example, an experiment was conducted to verify the detection accuracy of the ISH method for detecting the presence of another target substance, the genus Bacillus, in a sample as a model target.

[0063] For the RNA ISH method, we used RNA scope targeting a Bacillus-specific base sequence (SEQ ID NO: 2, Accession No. AB109633.1) to detect Bacillus species that may be present in tumors. TM The presence of the above-mentioned base sequence specific to the genus Bacillus was detected using the method (Wang F, et al., J Mol Diagn. 2012, PMID: 22166544).

[0064] When performing RNA ISH on the test sample, a control sample was also subjected to RNA ISH under the same conditions as the test sample. The control sample was amplified in E. coli using a Bacillus-specific base sequence (SEQ ID NO: 2, Accession No. AB109633.1).

[0065] Specifically, a Bacillus-specific sequence (SEQ ID NO: 2, Accession No. AB109633.1) was inserted into the pUCFa vector to generate the vector. This expression vector was then inserted into E. coli and the cells were cultured. After centrifugation at 6,000 × g for 10 minutes, the cells were fixed in formalin, embedded in 15 × 15 × 5 mm paraffin, and thinly sliced ​​to prepare tissue sections suitable for use as control samples in RNA ISH. RNA ISH was also performed on these control samples under the same conditions as the test samples. As a negative control, E. coli cells not transfected with the Bacillus-specific sequence (SEQ ID NO: 2, Accession No. AB109633.1) were used at the same concentration as the control samples, and RNA ISH was performed under the same conditions as the test samples.

[0066] The results are shown in Figure 2. Figure 2A shows the results of RNA ISH using a Bacillus-specific sequence as the target sequence. Specifically, the control sample showed a positive image (arrow) of the Bacillus-specific sequence (Figure 2A). No positive image was detected in the negative control sample (Figure 2B). This indicates that the Bacillus-specific sequence (SEQ ID NO: 2, AB109633.1) amplified in E. coli as the control sample was detected as a positive image by the ISH probe, demonstrating that the RNA ISH performed was performed accurately and without any experimental errors.

[0067] When detecting a target that is present in very small amounts in a sample, the present invention can demonstrate that the detection process is functioning correctly by detecting a control sample containing a positive control under the same conditions as the test sample, thereby providing a new method that can confirm that a target that is present in very small amounts in a sample can be detected with high sensitivity.

Claims

1. A positive control sample containing a non-pathogenic nucleic acid molecule containing the target base sequence detected by the ISH probe used in nucleic acid in situ hybridization (ISH), or a non-pathogenic peptide molecule or protein molecule containing the target amino acid sequence detected by immunostaining.

2. The sample according to claim 1, wherein the non-pathogenic nucleic acid molecule containing the target sequence detected by the ISH probe is introduced into a competent cell by a vector.

3. A sample according to claim 1 or 2, for use in nucleic acid in situ hybridization (ISH) by performing ISH on a positive control sample under the same conditions as on a test sample, thereby confirming the reaction of the detection system of the ISH method performed on the test sample.

4. The sample according to claim 1, wherein the non-pathogenic peptide molecule or non-pathogenic protein molecule containing the target sequence to be detected by immunostaining is a nucleic acid molecule encoding the peptide molecule or non-pathogenic protein molecule that has been introduced into a competent cell via a vector and expressed.

5. A sample according to claim 1 or 4, for use in an immunostaining method for peptide or protein molecules, by performing an immunostaining method on a positive control sample under the same conditions as on a test sample, thereby confirming the reaction of the detection system of the immunostaining method performed on the test sample.

6. In a nucleic acid in situ hybridization (ISH) method, (A) a test sample, and (B) a control sample containing a non-pathogenic nucleic acid molecule containing a target base sequence to be detected by an ISH probe, are subjected to the ISH method under the same conditions, thereby using (B) a control sample to evaluate the test sample and confirm the reaction of the detection system under the same conditions.

7. The method according to claim 6, wherein the ISH method for nucleic acids is an ISH method for RNA (RNA ISH method) or an ISH method for DNA (DNA ISH method).

8. The method according to claim 6 or 7, wherein the reaction confirmation of the detection system is to confirm that it is not affected by imperfections in the experimental procedure.

9. The method of claim 6 or 7, wherein (B) the non-pathogenic nucleic acid molecule containing the target base sequence in the control sample is obtained by artificial synthesis, viral synthesis, bacterial synthesis, or cellular synthesis.

10. In an immunostaining method for peptide molecules or proteins, (A) a test sample, and (B) a control sample expressing a non-pathogenic peptide molecule or non-pathogenic protein containing the target amino acid sequence detected by the antibody or antibody derivative used, are subjected to an immunostaining method under the same conditions as those for evaluating the test sample (A), thereby confirming the reaction of the detection system under the same conditions as those for evaluating the test sample (B).

11. The method according to claim 10, wherein the reaction confirmation of the detection system is to confirm that it is not affected by imperfections in the experimental procedure.

12. The method of claim 10 or 11, wherein (B) the non-pathogenic peptide molecule or non-pathogenic protein molecule containing the target amino acid sequence in the control sample is obtained by artificial synthesis, viral synthesis, bacterial synthesis, or cellular synthesis.

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