JP7378712B2 - Method for testing xenograft material for pathogen infection, method for producing test kit and xenograft material product evaluated for pathogen infection - Google Patents

Description

The present invention relates to a method for testing xenotransplant material for pathogen infection, which allows for easy testing of multiple types of pathogens, a test kit, and a method for producing a xenotransplant material product evaluated for pathogen infection.

Examples of animal species for xenotransplantation include mammals other than humans (eg, pigs, cows, primates, etc.), but these include the risk of infectious diseases. Pigs are the most studied animal species as xenograft donors, but they are susceptible to infectious diseases. For example, infectious diseases such as porcine circovirus-associated disease (PCVAD), porcine reproductive and respiratory syndrome (PRRS), porcine epidemic diarrhea (PED), and porcine fever have seriously affected pig populations worldwide. be.

Regarding pigs, which are the most studied xenograft donors, xenotransplant donors are DPF (Designated Pathogen Free) pigs, which are raised for medical purposes in a closed, well-controlled environment. In particular, there are many pigs that have eliminated the risk of zoonotic diseases.
DPF pigs have already been established in New Zealand, the United States, and other countries, and are being operated on a corporate basis (for example, Non-Patent Documents 1 and 2).
For example, in New Zealand, DPF pigs are tested for 27 types of pathogens, and 10 types of pathogens, 15 types of viruses, and 1 type of protozoa are tested regularly (once or four times a year depending on the target pathogen). ), and in the country, the main methods for detecting pathogens are ELISA and PCR (polymerase chain reaction; including real-time PCR).

Wynyard S, Nathu D, Garkavenko O, et al. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation 2014;21:309-323. Fishman JA. Infectious disease risks in xenotransplantation. Am J Transplant. 2018;18:1857-1864.

When testing for infection with multiple types of pathogens (e.g., multiple types of bacteria, viruses, protozoa) in animals for xenotransplantation (e.g., pigs, cows, primates, etc.) as described above, each pathogen is tested by PCR, etc. Testing requires effort, time, and cost, and is not practical for testing.There is a need for a method that can test for multiple types of pathogen infections in as few times as possible.

For example, when testing multiple types of pathogen infections using the PCR method, the melting temperature (Tm value) of each primer used in the same PCR reaction must be set at a predetermined value from the viewpoint of correlation with the annealing step in PCR. It is required to design to accommodate the difference.
However, as described above, as the number of types of pathogens to be tested increases, it becomes difficult to design primers so that the melting temperatures (Tm values) of each primer used in the same PCR reaction fall within a predetermined difference.

The present invention has been made in view of the actual state of the prior art, and provides a method, a test kit, and a test kit for testing pathogen infection in xenograft materials that can easily test for multiple types of pathogens. The purpose is to provide an evaluated method for producing xenograft material products.

In testing for pathogen infection in xenograft materials, the species detected for viruses and protozoa is important, but once infected with bacteria, regardless of the type, the xenograft material should be avoided as a transplant material. , the present inventors paid attention to this.
Therefore, the present inventors have discovered that the test can be simplified by using primers for common base sequences between at least two types of bacteria and focusing on detecting the presence or absence of bacteria rather than identifying the species. The present invention has been completed based on the above findings.
That is, the present invention is as follows.

<1> The step includes a step of genetically testing infection of pathogens including two or more types of bacteria and viruses or protozoa, and in the genetic testing of the two or more types of bacteria, a primer for a common base sequence between at least two types of bacteria. A method for testing xenograft material for pathogen infection using
<2> The method according to <1>, wherein the common base sequence is a sequence included in a conserved region of bacterial 16S rRNA.
<3> The conserved region is a conserved region common to at least 16S rRNA of each of Leptospira, Mycoplasma, Campylobacter, Yersinia, E. coli, and Salmonella. the method of.
<4> The method according to <2>, wherein the storage area is an area including V3 and V4 areas.
<5> The method according to any one of <1> to <4>, wherein one pair of primers is used in the genetic testing of the two or more types of bacteria.
<6> The genetic testing for infection with two or more types of bacteria and the genetic testing for viral or protozoan infection are performed by PCR under the same thermal cycle conditions, and the difference in melting temperature of all primers used is 15%. ℃ or less, the method according to any one of <1> to <5>.
<7> The method according to any one of <1> to <6>, wherein the pathogen infection is an infection that causes a zoonotic disease.
<8> The step of genetically testing for infection by a virus is a step of testing for infection by five or more types of viruses by a PCR method using a primer pair that selectively binds to the nucleic acid of each virus, or The step of genetically testing for infection by two or more types of protozoa is a step of testing for infection by two or more types of protozoa by a PCR method using a primer pair that selectively binds to the nucleic acid of each protozoa, <1> to <7 ＞The method according to any one of ＞.
<9>
The method according to any one of claims 1 to 8, wherein the xenograft material is a porcine-derived xenograft material.
<10> The virus includes porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus type H1N1, porcine lymphotropic herpesvirus, porcine cytomegalovirus, and hepatitis E virus, or the protozoan The method according to any one of <1> to <9>, including Toxoplasma gondii.
<11> A test kit comprising a primer for a common base sequence between at least two types of bacteria and a primer that selectively binds to the nucleic acid of at least one type of virus or protozoa.
<12> A method for producing a material product for xenotransplantation that has been evaluated for pathogen infection, comprising the step of implementing the method according to any one of <1> to <10> above.

According to the present invention, there are provided a method for testing pathogen infection of a xenotransplant material, which allows multiple types of pathogen tests to be easily performed, a test kit, and a method for producing a xenotransplant material product evaluated for pathogen infection. obtain.

FIG. 1 is a diagram showing the results of PCR product electrophoresis of 15 types of viruses to be detected by Standard PCR (FIG. 1(A)) and Nested PCR (FIG. 1(B)). FIG. 3 is a diagram showing pathogen detection results obtained by evaluating a PCR test system using clinical specimens. FIG. 2 is a diagram showing the results of cytomegalovirus PCR product electrophoresis using specific primers.

Embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the purpose of the present invention. .

≪Method for testing xenograft materials for pathogen infection≫
A first aspect of the present invention includes a step of genetically testing for infection with a pathogen including two or more types of bacteria and a virus or a protozoan, and in the genetic testing of the two or more types of bacteria, an infection between at least two types of bacteria is detected. This is a method for testing pathogen infection of xenograft materials using primers for the common base sequence of .
The first aspect is to perform similar inspection work using different primer sets (hereinafter also referred to as "primer pairs"; a pair of Forward primer (F primer) and Reverse primer (R primer)) for each individual pathogen. Duplicate work can at least be reduced by using primers directed to common base sequences between at least two types of bacteria.
It is preferable that the step of genetically testing the two or more types of bacteria and the step of genetically testing the virus or protozoa are both performed by PCR.

In the present invention, the PCR method may be a standard PCR method or not. It may or may not be the so-called RT-PCR method, in which complementary cDNA is synthesized and then PCR is performed.
Furthermore, the PCR method may or may not be real-time PCR from the viewpoint of improving quantitative performance.
Furthermore, the PCR method may or may not be a modified method such as nested PCR from the viewpoint of improving specificity and yield.

The material for xenotransplantation to be tested includes materials derived from any animal for xenotransplantation, and the animals for xenotransplantation include pigs, cows, sheep, primates (e.g., chimpanzees, monkeys), etc. , pigs are preferred.
Examples of the material for xenotransplantation include organs (liver, kidney, pancreas, heart, etc.), blood, urine, sweat, etc., with organs or blood being preferred.
There are no particular restrictions on the animals used for xenotransplantation, and even if they are wild-type, animals raised for medical purposes in a closed, well-controlled environment that eliminate the risk of infectious diseases, especially zoonotic diseases. (For example, in the case of pigs, DPF pigs) may be used. Examples of the above-mentioned DPF pigs include pigs bred by the UiR method.

In the first aspect, the types of bacteria to be genetically tested are not particularly limited as long as they are 2 or more types, preferably 5 or more types, more preferably 10 or more types, even more preferably 15 or more types.
The upper limit of the types of bacteria to be genetically tested is not particularly limited, but is, for example, 100 types or less of bacteria, or 80 types or less of bacteria.
Of all the types of bacteria to be genetically tested, the types of bacteria to be genetically tested using primers for common base sequences between the at least two types of bacteria preferably account for 50% or more, and preferably account for 70% or more. is more preferable, and even more preferably accounts for 90% or more.
The primers used in the genetic testing of two or more types of bacteria are not particularly limited as long as they contain a sequence corresponding to the common base sequence between at least two types of bacteria, and for example, they may contain any other sequences in addition to the above sequences. obtain. The base length of the primer is not particularly limited as long as it can function as a primer by selectively binding to a common base sequence between at least two types of bacteria; for example, 10 to 50 The length of the base can be mentioned, and the length is preferably 12 to 40 bases, and more preferably 15 to 30 bases.
The primers used in the genetic testing of the two or more types of bacteria mentioned above may be two or more pairs (for example, 2 pairs, 3 pairs, 4 pairs, ... 10 pairs), but the complexity of the test From the viewpoint of avoiding problems, it is preferable to use one pair of primers.
The primers used in the genetic testing of the above two or more types of bacteria include primers for the common base sequence between at least two types of bacteria, as well as primers that detect other bacteria (for example, bacteria that do not have the above common base sequence). Therefore, primers that selectively bind to the nucleic acids of the other bacteria may or may not be used together.

The Tm value of the primers used in genetic testing of the above two or more types of bacteria is preferably 50°C or higher, and 55°C or higher, from the viewpoint of avoiding non-specific annealing with genomic genes (for example, pig genomic genes). It is more preferable that it is above.
Further, the upper limit of the Tm value of the primer is not particularly limited, but is, for example, 80°C or lower, preferably 75°C or lower.
The primer for the common base sequence between at least two types of bacteria is preferably a primer for a common base sequence between three or more types of bacteria, more preferably a primer for a common base sequence between five or more types of bacteria, and a primer for a common base sequence between at least 5 types of bacteria is preferable. Primers for common base sequences among bacteria are more preferred, primers for common base sequences for 15 or more types of bacteria are particularly preferred, and primers for common base sequences for 20 or more types of bacteria are most preferred.
The upper limit is not particularly limited, but is, for example, 100 types or less of bacteria, or 80 types or less of bacteria.

16S ribosomal RNA (rRNA) can be shared by all bacteria. 16S rRNA has regions that are highly conserved across bacterial species (eg, conserved regions C1 to C9).
The above-mentioned common base sequence is preferably a sequence included in the conserved region of bacterial 16S rRNA from the viewpoint of achieving the effects of the present invention more reliably.
From the viewpoint of high conservation among a plurality of bacteria, the conserved region is preferably a region containing the V3 and V4 variable regions (or a plurality of sandwiched regions).
The following are examples of primer pairs for regions containing the V3 and V4 variable regions, but the primer pairs in the present invention are not limited to these.
341F: 5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO: 1)
805R: 5'-GACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 2)

The test target, which is a pathogen containing two or more types of bacteria and a virus or a protozoa, is preferably a pathogen containing two or more types of bacteria and both a virus and a protozoa.
Furthermore, the test target may or may not contain fungi such as Trichophyton and other dermatophytes.
Bacteria, viruses, and protozoa to be tested include bacteria, viruses, and protozoa that cause zoonotic diseases, zoonotic diseases (e.g., swine infections), or human infectious diseases; Bacteria, viruses, and protozoa that cause infectious diseases are preferred.
Examples of the at least two types of bacteria to be tested include at least two types of bacteria selected from the following specific examples.
Specific examples: Yersinia bacterium, Bronchiseptica bacillus, Clostridium, tuberculosis (human type, bovine type, avian type), Salmonella, Escherichia coli, Bacillus anthrax, Erysipelas swine, Pasteurella, Shigella swine, Haemophilus, Staphylococcus, Brucella , Hemopartonella, Mycoplasma, Listeria monocytogenes, Actinobacillus, Streptococcus, Pseudomonas aeruginosa, Porcine actinomyces, Campylobacter, Actinobacillus, Chlamydia, Coxiella, Lawsonia, Leptospira (Leptopira), Porcine perosthrozoon.

The at least two types of bacteria are preferably at least two types selected from the group consisting of Leptospira, Mycoplasma, Campylobacter, Yersinia, E. coli, and Salmonella, and the storage region is Preferably, the conserved region is at least common to 16S rRNA of Leptospira, Mycoplasma, Campylobacter, Yersinia, E. coli, and Salmonella.

Specifically, the viruses to be tested include porcine circovirus type 1, porcine circovirus type 2, porcine lymphotropic herpesvirus 1, porcine lymphotropic herpesvirus 2, porcine cytomegalovirus, and encephalomyocarditis. Virus, porcine parvovirus, porcine rotavirus A, porcine rotavirus B American type, porcine rotavirus B Vietnam type, porcine rotavirus C, mammalian reovirus, porcine enterovirus, porcine hemagglutinating encephalomyelitis virus, swine reproduction and respiration Disorder syndrome virus, hepatitis E virus, swine infectious gastroenteritis virus, swine influenza virus type H1N1, swine influenza virus type H3N2, swine respiratory coronavirus, swine fever virus, swine pox virus, African ton fever virus, Aujeski's disease Virus, getavirus, Japanese encephalitis virus, swine epidemic diarrhea virus, swine vesicular disease virus, swine vesicle virus, vesicular stomatitis virus new jersey, vesicular stomatitis virus indiana, foot and mouth disease virus, rabies virus 1, rabies virus 2 , rabies virus 3, rabies virus 4, rabies virus 5, rabies virus 6, rabies virus 7, porcine adenovirus, porcine astrovirus 1, porcine astrovirus 2, porcine astrovirus 3, porcine astrovirus 4, porcine astrovirus 5, Menangle virus, Nipah virus, Hantavirus, Eastern equine encephalitis virus 1, Eastern equine encephalitis virus 2, Eastern equine encephalitis virus 3, Eastern equine encephalitis virus 4, Western equine encephalitis virus, Venezuelan equine encephalitis virus, Borna virus, Apoi virus, Poly Omavirus, bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, bovine viral diarrhea virus type 3, bovine infectious rhinobronchitis virus, porcine torquetenovirus type 1, porcine torquetenovirus type 2, support Virus type 3, sapovirus type 6, sapovirus type 7a, sapovirus type 7b, sapovirus type 7c, sapovirus type 8, sapovirus type 9, sapovirus type 10, norovirus type A, norovirus type B, porcine burravirus , calicivirus, spumavirus, porcine gamma herpesvirus, and porcine endogenous retrourus, but from the viewpoint of low pathogenicity, porcine endogenous retrourus may not be included in the test target.
The viruses to be tested preferably include at least porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus type H1N1, porcine lymphotropic herpesvirus, porcine cytomegalovirus, and hepatitis E virus.

Specifically, the protozoa to be tested include Toxoplasma gondii, Coccidium, Palantidium, Cryptosporidium, Sarcocystis, Babesia, Trypanosoma spp., Ascaris porcine, Toxocara, Echinococcus, Rhododendron nematode, Leech-like talcocephalus, Examples include lungworm, Strongyloides, Taenia solium, ciliates, hairy nematodes, Taenia trichoides, and other ectoparasites, and it is preferable that at least Toxoplasma gondii is included as the protozoa to be tested.

The step of genetically testing the virus or protozoan is performed by PCR using a primer pair that selectively (preferably specifically) binds to the nucleic acid (DNA, RNA, etc.) of at least one type of virus or protozoan. Preferably, it is a process.
The primer that selectively binds to the nucleic acid of each of the at least one type of virus or protozoa is not particularly limited, and may, for example, contain any other sequence in addition to the selectively binding sequence. The base length of the primer is not particularly limited as long as it can function as a primer capable of selectively binding to the nucleic acid of the at least one type of virus or protozoa. Examples include a length of 50 bases, preferably a length of 12 to 40 bases, and more preferably a length of 15 to 30 bases.
The Tm value of the primer is preferably 50° C. or higher, more preferably 55° C. or higher, from the viewpoint of avoiding non-specific annealing with a genomic gene (for example, a pig genomic gene).
Further, the upper limit of the Tm value of the primer is not particularly limited, but is, for example, 80°C or lower, preferably 75°C or lower.
Further, the melting temperature difference of all the primers used for the above-mentioned bacterial infection test and the above-mentioned viral or protozoan infection test is 25°C or less (more preferably 20°C or less, still more preferably 15°C or less, particularly preferably 12° C. or lower) is preferable.
The smaller the difference in melting temperature between all the primers, the better, and there is no particular restriction on the lower limit of the difference in melting temperature. ℃ or higher, and ideally 0°C.

The step of genetically testing for infection by at least one type of virus is performed to detect infection by 5 or more types of viruses (preferably 10 or more types, more preferably 20 or more types, even more preferably 25 or more types). Preferably, the step is a step of testing by PCR using a primer pair that selectively binds to nucleic acids.
There is no particular limit to the upper limit of virus types, but for example, it is 100 types or less, 50 types or less.
Further, the step of genetically testing for infection by at least one type of protozoa may include genetically testing for infection by two or more types (preferably three or more types) of protozoa using a primer pair that selectively binds to the nucleic acid of each protozoa. Preferably, this is a step of testing by PCR method.
The upper limit of the types of protozoa is not particularly limited, but is, for example, 10 or less types, 5 or less types.

The following are examples of primer pairs that selectively bind to viral nucleic acids, but the primer pairs in the present invention are not limited to these.
(Primer pair for porcine reproductive and respiratory syndrome virus)
F: 5'-CACTGTGGAGTTTAGTTTGC-3' (SEQ ID NO: 3)
R: 5'-CACACGGTCGCCCTAATTGAATA-3' (SEQ ID NO: 4)
(Primer pair for porcine circovirus (type 2))
F: 5'-CCAGGAGGGCGTTGTGACT-3' (SEQ ID NO: 5)
R: 5'-CGCTACCGTTGGAGAAGGAA-3' (SEQ ID NO: 6)
(Primer pair for swine influenza virus (H1N1 type))
F: 5'-AGGATTTGTGTTCACGCTCA-3' (SEQ ID NO: 7)
R: 5'-GCATTTTGGACAAAGCGTCT-3' (SEQ ID NO: 8)
(Primer pair for porcine lymphotropic herpesvirus (types 1 and 2))
F: 5'-AGGACCTGCATAAGTATCCTCAA-3' (SEQ ID NO: 9)
R: 5'-CAGGGCCAGAACTAATCAAAA-3' (SEQ ID NO: 10)
(Primer pair for porcine cytomegalovirus)
F: 5'-AATGCGTTTTACGGCTTCAC-3' (SEQ ID NO: 11)
R: 5'-ACGCCGCTAGAGGGAGAC-3' (SEQ ID NO: 12)
(Primer pair for hepatitis E virus (ORF1) (see Non-Patent Document 1))
F: 5'-CTGGCATYACTACTGCYATTGAGC-3' (SEQ ID NO: 13)
R: 5'-CCATCRARRCAGTAAGTGCGGTC-3' (SEQ ID NO: 14)

The following are examples of primer pairs that selectively bind to protozoan nucleic acids, but the primer pairs in the present invention are not limited to these.
(Primer pair against Toxoplasma gondii)
F: 5'-CTAGGGCACCCTTACTGCAA-3' (SEQ ID NO: 15)
R: 5'-CTGCTGGATCTCTTCCCTTG-3' (SEQ ID NO: 16)

In the step of genetically testing the two or more types of bacteria and the step of genetically testing the virus or protozoa, the detection of infection by the two or more types of bacteria, and the detection of infection by the virus or protozoa include the two or more types of bacteria. There is no particular restriction as long as the nucleic acids of viruses or protozoa can be detected statistically significantly. For example, the nucleic acid is detected 1.1 times or more, preferably 1.2 times or more, and more preferably 1.5 times or more compared to a control.
The upper limit for detection of the nucleic acid is not particularly limited, but may be, for example, 10 times or less.
The above-mentioned control may be a statistical value or a value previously collected from a group of healthy animals (e.g., healthy pigs), even if it is a measurement value obtained from an animal (e.g., a pig) before virus infection. It may be a range.

The detection of infection may or may not be based on the intensity of the label (e.g., absorbance, enzyme label intensity, fluorescence intensity, ultraviolet intensity, radiation intensity, etc.) measured in the labeling substance (e.g., labeled antibody).

In the step of genetically testing two or more types of bacteria and the step of genetically testing the virus or protozoa, there are no particular restrictions on the PCR thermal cycle conditions (e.g., temperature, time, number of cycles, etc.) Taking common technical knowledge into account, we set the temperature and time appropriately for the denaturation reaction that converts double-stranded nucleic acids into single strands, annealing reaction, and nucleic acid elongation reaction by nucleic acid synthases such as DNA polymerase, and conducted these reactions over several dozen times. This can be carried out by repeating the cycle.
Denaturation reaction conditions are not particularly limited, and include, for example, a reaction in which the target nucleic acid is heated at 90° C. to 100° C. for several tens of seconds to several minutes.
The annealing reaction is a reaction in which the primer and the target nucleic acid are placed under conditions such that the primer and the target nucleic acid hybridize, but is not particularly limited. C. (preferably 52.degree. C. to 65.degree. C.) for several tens of seconds to several minutes.

The nucleic acid elongation reaction is not particularly limited, and can include, for example, a reaction using a nucleic acid synthase such as DNA polymerase and carried out at a temperature suitable for the enzymatic activity of the nucleic acid synthase.

It is preferable that the step of genetically testing two or more types of bacteria and the step of genetically testing the virus or protozoa are performed simultaneously from the viewpoint of conducting the tests as few times as possible.
From the viewpoint of performing the test as few times as possible, it is preferable that the genetic test for infection with two or more types of bacteria and the genetic test for virus or protozoan infection are performed by PCR under the same thermal cycle conditions. Further, the melting temperature difference of all the primers used for the above-mentioned bacterial infection test and the above-mentioned viral or protozoan infection test is 25°C or less (more preferably 20°C or less, still more preferably 15°C or less, particularly preferably 12° C. or lower) is preferable.
The smaller the difference in melting temperature between all the primers, the better, and there is no particular restriction on the lower limit of the difference in melting temperature. ℃ or higher, and ideally 0°C.
There are no particular restrictions on the timing, frequency, etc. of implementing the testing method according to the first aspect.

≪Test kit≫
A second aspect of the present invention is a test kit that includes a primer for a common base sequence between at least two types of bacteria and a primer that selectively binds to the nucleic acid of at least one type of virus or protozoa.
Specific examples and preferred examples of the primer for the common base sequence between the at least two types of bacteria include those similar to the primers described above in the first embodiment.
Specific and preferred examples of primers that selectively bind to at least one type of virus or protozoan nucleic acid include those similar to the primers described above in the first embodiment.
The test kit according to the second aspect may or may not contain any components for performing PCR, such as DNA polymerase, dNTP, and PCR reaction buffer.
Furthermore, the test kit according to the second aspect may also include a container that may contain each of the above components.
In the test kit according to the second aspect, the primer for the common base sequence between the at least two types of bacteria and the primer that selectively binds to the nucleic acid of the at least one type of virus or protozoa are arranged on an arbitrary substrate (e.g. It may or may not be a kit fixed (preferably in an array) on a plastic substrate).

≪Method for creating xenotransplant material products evaluated for pathogen infection≫
A third aspect of the present invention is a method for producing a material product for xenotransplantation evaluated for pathogen infection, which includes a step of implementing the testing method according to the first aspect.
Materials in which infection with pathogens including bacteria, viruses, or protozoa are detected through the above steps are evaluated as unusable as material products for xenotransplantation; Materials that were previously unavailable may be evaluated for use as xenograft material products.
Examples of material products for xenotransplantation include organ products (liver, kidney, pancreas, heart, etc.), blood products, urine products, sweat products, etc., with organ products or blood products being preferred.
The material product for xenotransplantation obtained according to the second aspect has a reduced risk of pathogen infection and can be suitably used for xenotransplantation.

Examples of the present invention will be shown below to further specifically explain the present invention, but the present invention is not limited to these, and various applications can be made without departing from the technical idea of the present invention. It is.

<1. Establishment of a pathogen detection system using PCR>
(1.1. Primer)

For viruses and protozoa, primers specific to each pathogen shown in Table 1 above were used. The Tm value of each of the above primers falls within the range of 60 to 75°C.
In the above table, "reaction composition and conditions of PCR used" corresponds to Tables 2 and 3 below.
In the above table, the F and R primers used for 2nd PCR in nested PCR to improve specificity and yield are indicated as nested F and R, respectively.
The "positive control" in the above table is as follows.
A: Nucleic acid extracted from the target pathogen B: Nucleic acid extracted from Nisseiken PED live vaccine (manufactured by Nisseiken Co., Ltd.) C: Nucleic acid extracted from swine stillbirth three-type live vaccine (manufactured by Microbial Science Institute Co., Ltd.) D: Artificially synthesized DNA

All bacteria were targeted by using universal primers (SEQ ID NO: 1 and 2) targeting the conserved region of the 16S rRNA gene.

The above-mentioned specific primer used in this example was adopted under the condition that it satisfied the following two conditions.
Condition 1: Broadly cover multiple strains of the target pathogen, not a single strain. Obtain the entire genome of the target pathogen from Genbank and around 50 sequences of genes targeted by primers, and perform alignment using CLC Main Workbench (manufactured by QIAGEN). ), it was confirmed that the primers contained mismatches within three bases at most for all sequences.
In addition, all registration data were obtained for pathogens with 50 or less registrations in Genbank.
Condition 2: High specificity for the target pathogen Using the Basic Local Alignment Search Tool (BLAST (registered trademark)) (manufactured by National Institutes of Health), the primers are homologous to genes other than the target pathogen and pig genes. It was confirmed that no
As a general rule, previously published specific primers are adopted with priority, and if the above two conditions are not met, a consensus sequence in a region that is highly conserved between strains is selected from the above alignment, and the website The primers were designed by introducing them into Universal Probe Library Assay Design Center (manufactured by Roche), which is a base primer creation software.

(1.2. PCR positive control)
As the positive controls shown in Table 1 above, any one was used according to the following priority.
(1) Nucleic acid extracted from the target pathogen (2) Nucleic acid extracted from a commercially available live vaccine against the target pathogen (3) Artificially synthesized DNA

The viruses for which the nucleic acids of target pathogens were used as positive controls were porcine reproductive and respiratory syndrome virus, porcine circovirus (type 2), and swine influenza virus (type H1N1), all of which are RNA viruses.
To use as a positive control, RNA was extracted from the plasma of a pig that a veterinarian at a general farm judged to be potentially infected with the pathogen using the QIAamp Viral RNA kit (manufactured by QIAGEN) according to the protocol included with the kit. was extracted. However, carrier RNA was not used during RNA extraction.

cDNA was synthesized using SuperScript (registered trademark) II Reverse Transcriptase (manufactured by Thermo Fisher Scientific). A 12 μl reaction containing 1.5 μl of Random Primers (manufactured by Thermo Fisher Scientific), 4 μl of 2.5 mM dNTP Mix, 1 μl of RNA (250 ng/μl), and 5.5 μl of Nuclease Free Water (NFW). A liquid was prepared. The mixture was allowed to stand at 65°C for 5 minutes and immediately transferred to ice. Subsequent operations were performed in accordance with the protocol included with the kit. Using this cDNA, PCR amplification was performed using the method described previously (Inoue R, Tsukahara T, Sunaba C, et al.: 2007, Simple and rapid detection of the porcine reproductive and respiratory syndrome virus from pig whole blood using filter paper J Virol Methods 141:102-106., Olvera A, Sibila M, Calsamiglia M, et al.: 2004, Comparison of porcine circovirus type 2 loaded in serum quantified by a real time PCR in postsweaning multisystemic wasting syndrome and porcine dermatitis and After confirming the base sequence by sequence analysis, this cDNA was used as a positive control.
For swine influenza virus (H1N1 type), 5 μl of LightCycler (registered trademark) 480 Probes Master, 0.1 μl of probe #104 (manufactured by Roche), 0.4 μl of each of the specific primer sets shown in Table 1 at 10 μM, NFW3. A total of 10 μl of the reaction solution, 1 μl of the template nucleic acid and 1 μl of the template nucleic acid, were mixed, and the rest was carried out in the same manner as for the other pathogens described above.

For bacteria, DNA extracted from Competent Quick DH5α (Green Cap) (manufactured by TOYOBO) using Quick Gene DNA tissue kit S (manufactured by KURABO) was used.
RNA extraction and cDNA production from commercially available live vaccines were performed in the same manner as above.

Artificial synthetic DNA was produced by adding approximately 30 bp above and below the target region of the consensus sequence primer described in the above (1.1. Primer) section as gBlocks (registered trademark) Gene Fragments (manufactured by INTEGRATED DNA TECHNOLOGIES). Synthesized. Each pathogen (apoivirus, hepatitis E virus (ORF1), hepatitis E virus (ORF2), bovine viral diarrhea virus (type 1), bovine viral diarrhea virus (type 2), bovine viral diarrhea virus (type 3) ), rabies virus (type 4), rabies virus (type 5), rabies virus (type 6), rabies virus (type 7), foot-and-mouth disease virus, respiratory coronavirus, sapovirus (type 3), sapovirus (type 6) ), sapovirus (type 7A), sapovirus (type 7B), sapovirus (type 7C), sapovirus (type 8), sapovirus (type 9), sapovirus (type 10), vesicular stomatitis virus ( New Jersey type), Western equine encephalitis virus, hemagglutinating encephalomyelitis virus, Ton cholera virus, Nipah virus, encephalomyocarditis virus, Norovirus (type A), Norovirus (type B), Porcine astrovirus (type 1), Porcine Astro Virus (type 3), swine astrovirus (type 5), swine influenza virus (H1N1 type), swine influenza virus (H3N2 type), swine enterovirus (type B), swine vesicle virus, swine vesicular disease virus, swine contagious Gastroenteritis virus, porcine torquetenovirus (type 1), porcine torquetenovirus (type 2), rubula virus, porcine rotavirus (type A), porcine rotavirus (American type B), porcine rotavirus (Vietnamese type B) , porcine rotavirus (type C), mammalian reovirus, bornavirus, menangle virus, African tonkolera virus, bovine infectious rhinotracheitis virus, Aujeski's disease virus, porcine adenovirus, porcine cytomegalovirus, porcine circovirus (type 1), porcine parvovirus, porcine poxvirus, porcine lymphotropic herpesvirus (types 1 and 2), and Toxoplasma gondii). It was defined as an area.

(1.3. Flow of establishing detection system)
As an evaluation of the detection system, it was confirmed that conditions 1 to 3 below were satisfied.
Condition 1: PCR is performed using a positive control gene suspended in NFW as a positive control and NFW as a negative control, and the target product is amplified only with the positive control, and non-specific products are not amplified with the negative control.
Condition 2: Only the gene of interest should be amplified even in a positive control mixture (in NFW) in which positive control genes of around 10 pathogens are mixed in equal amounts.
Condition 3: A positive control gene is mixed with cDNA derived from pig pancreas, which has been confirmed in advance that the target gene will not be amplified, and PCR is not inhibited by the pig-derived nucleic acid.

(1.4. Virus detection by Standard PCR)
Pathogens were detected by standard PCR using LifeECO (manufactured by Nippon Genetics). Two types of PCR reaction compositions used in this study are listed in Table 2 below, and six types of reaction conditions are listed in Table 3 below. After amplification, electrophoresis was performed on a 1.5% agarose gel using Tris-boric acid-EDTA buffer stock solution (manufactured by Nacalai Tesque), and it was confirmed that the size matched the target size.

(1.5. Virus detection by nested PCR)
LifeECO was used in the same manner as in the above section (1.4. Detection of virus by Standard PCR). The reaction composition was the same for both 1st PCR and 2nd PCR as shown in Table 2 above. As the template nucleic acid for 2nd PCR, the 1st PCR product was used as it was without dilution or purification. The reaction conditions were the same for both 1st PCR and 2nd PCR, and were performed under the conditions shown in Table 3 above.
Confirmation of the amplified product by electrophoresis was carried out in the same manner as in the above section (1.4. Detection of virus by standard PCR).

(1.6. Detection of viruses and protozoa by real-time PCR)
Light Cycler (registered trademark) 480 (manufactured by Roche) was used. A total of 10 μl of the reaction solution was mixed: 5 μl of SYBR (registered trademark) Premix Ex TaqII (Tli RNase Plus) (manufactured by TAKARA), 0.2 μl each of the 10 μM primer set, 3.6 μl of NFW, and 1 μl of template nucleic acid. The reaction was performed in duplicate. The reaction conditions for each pathogen are shown in Table 3 above.
After performing melting curve analysis, the PCR products were electrophoresed using a 2% agarose gel (Tris-borate-EDTA) and confirmed to match the desired size.

(1.7. Bacteria detection)
For bacterial detection, 341F:5'-CCTACGGGNGGCWGCAG-3' (Tm value 68°C; SEQ ID NO: 1), 805R:5' were used as primers targeting the highly conserved region of the 16s rRNA gene (v3-v4 region). -GACTACHVGGGTATCTAATC-3' (Tm value 56.5°C; SEQ ID NO: 2) was used (Klindworth A et al., 2013).
PCR was performed using a total of 25 μl of the reaction solution, including 12.5 μl of KAPA HiFi Hot Start Ready Mix (manufactured by Nippon Genetics), 5 μl of each 0.2 μM primer set, and 2.5 μl of DNA. After initial denaturation at 95°C for 3 minutes, 35 cycles of heat denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension reaction at 72°C for 30 seconds were performed. A final extension reaction was then performed at 72° C. for 5 minutes.
Confirmation of the amplified product by electrophoresis was performed in the same manner as in the above section (1.4. Detection of virus by standard PCR).

(1.8. Measurement of detection limit copy number)
((Preparation of plasmid for positive control))
For detection systems that use nucleic acid extracted from the target pathogen or a commercially available live vaccine against the target pathogen as a positive control, this should be used to determine the exact copy number of the positive control DNA. The containing plasmid was obtained by the following method.
The PCR product obtained by the above method was purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (manufactured by Promega).
The PCR product was mixed with an equal amount of Membrane Binding Solution, and the entire amount was added to Econo Spin (registered trademark) for DNA (manufactured by Epoch Biolabs). After standing at room temperature for 1 minute, centrifugation was performed at room temperature and 16,000×g for 1 minute. The filtrate was removed, 400 μl of Membrane Wash Solution was added, and centrifugation was performed under the same conditions as the previous time. The filtrate was removed again, 400 μl of Membrane Wash Solution was added, and the mixture was centrifuged at 16,000×g for 5 minutes at room temperature. Another tube was set in the column, 30 μl of NFW was added, and the tube was left standing at room temperature for 1 minute. Centrifugation was performed at 16,000×g for 1 minute at room temperature.
Thereafter, it was integrated into a cloning vector provided with the kit using TOPO TA (registered trademark) Cloning (registered trademark) kit (manufactured by Thermo Fisher Scientific). 2 μl of the vector was transformed into Competent Quick DH5α (Green Cap) (manufactured by TOYOBO) by heat shock method, and the transformant was inoculated into LB medium.
Colony PCR was performed on the grown colonies to select vectors into which the insert had been inserted. For colony PCR, GoTaq (registered trademark) Green Master Mix (manufactured by Promega) was used, Master Mix 12.5 μl, and M13 included in TOPO TA (registered trademark) Cloning (registered trademark) kit (manufactured by Thermo Fisher Scientific). Forward Primer and A total of 25 μl of reaction solution was prepared, including 0.5 μl each of M13 Reverse Primer (10 μM) and 11.5 μl of NFW. A colony was lightly touched with a 10 μl pipette tip, and the tip was added to the reaction solution. After initial denaturation at 94°C for 2 minutes, 30 cycles of heat denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, and extension reaction at 72°C for 30 seconds were performed. Electrophoresis was performed in the same manner as in the above section (1.4. Detection of virus by Standard PCR).
Colonies in which a band of the desired size was confirmed were inoculated into 6 ml of LB liquid medium containing 50 μg/ml ampicillin, and cultured at 37° C. overnight.
Plasmid extraction was performed using Mini Plus (registered trademark) Plasmid DNA Extraction System (manufactured by VIOGENE) according to the protocol included with the kit. The concentration of the plasmid was measured using a NanoDrop (registered trademark) 1000 Spectrophotometer (manufactured by Thermo Fisher Scientific), and the copy number was calculated.

((Measurement of detection limit copy number))
The detection limit copy number was measured only for selected viruses that exist or may exist in Japan.
Six levels of samples were prepared in a 10-fold dilution series so that the maximum concentration was approximately 1 to 9 × 10 ⁵ copies/μl and the minimum concentration was approximately 1 to 9 copies/μl, and this was used as described above (1.4. Detection of viruses and protozoa) to (1.6. Detection of viruses and protozoa by real-time PCR) above, PCR was performed under the conditions for each pathogen.
Only in real-time PCR, the reaction was performed in duplicate, but when determining the detection limit, if amplification of the target product was confirmed in one of the two replicates at the dilution stage that was the limit, it was determined to be positive.
Regarding real-time PCR, PCR efficiency was calculated using the following formula. Note that the slope included in the following equation refers to the slope of a graph in which the vertical axis is the cycle number and the horizontal axis is the Log copy number.

<2. Evaluation of PCR test system using clinical specimens>
(2.1. Clinical specimen)
As clinical specimens, a specimen considered to have a high degree of cleanliness (clean sample) and a specimen having a low degree of cleanliness, that is, a specimen having a high possibility of being infected with a pathogen (infected sample) were prepared. As clean samples, the liver, kidney, pancreas, and blood of two 21-day-old piglets raised as DPF candidate pigs by the U-iR method (Clean samples 1 and 2) were used. The infected samples used were the liver, kidney, lung, and lymph node of a pig that was examined and dissected by a veterinarian on suspicion of infection at a general farm.

(2.2. RNA extraction from organs)
50 mg of the organ was chopped with ophthalmic scissors, 1 ml of TRIzol (registered trademark) LS Reagent (manufactured by Life Technologies) was added, and then homogenized using Tissue Ruptor II (manufactured by QIAGEN). After adding 200 μl of chloroform (manufactured by Wako) and vortexing for 15 seconds, the mixture was incubated at room temperature for 2 minutes. Centrifugation was performed at room temperature and 12,000×g for 15 minutes, and 350 μl of the transparent upper layer (aqueous layer) after centrifugation was collected into another tube. RNA was purified from the collected upper layer using RNeasy (registered trademark) Mini kit (manufactured by QIAGEN).

After adding 70% (v/v) ethanol in an amount equal to that of the collected upper layer and mixing by inversion, the entire amount of the mixed solution was applied to the column included in the kit. Centrifugation was performed at 8,000×g for 15 seconds at room temperature, and the filtrate was discarded. 350 μl of Buffer RW1 was added to the column, centrifugation was performed under the same conditions as before, and the filtrate was discarded. For the DNase treatment, RNase-Free DNase Set (manufactured by QIAGEN) was used. 75 μl of a solution in which DNase I and Buffer RDD included in the kit were suspended at a ratio of 1:7 was added, and the mixture was allowed to stand at room temperature for 8 minutes.
350 μl of RW1 was added, centrifuged under the same conditions as before, and the filtrate was discarded. 500 μl of Buffer RPE was added, centrifuged under the same conditions as before, and the filtrate was discarded. 500 μl of Buffer RPE was added again, centrifuged at 8,000×g for 2 minutes at room temperature, and the filtrate was discarded. Nothing was added to the column and the column was centrifuged at 20,000 xg for 1 minute at room temperature. Another tube was set in the column, 50 μl of RNase Free Water was added, and the tube was allowed to stand for 1 minute, followed by centrifugation at 20,000×g for 1 minute at room temperature.

(2.3. RNA extraction from blood)
After adding 750 μl of TRIzol (registered trademark) LS Reagent (manufactured by Life Technologies) to 250 μl of heparinized blood, the mixture was vortexed and allowed to stand at room temperature for 5 minutes. The subsequent operations were performed in the same manner as in the above section (2.2. RNA extraction from organs) except for the following steps.
For RNA extraction from blood, chloroform was added and 560 μl of the upper layer (aqueous layer) was collected after centrifugation, without DNase treatment, and in place of the second wash of Buffer RPE, 80% (v/ v) Washing with 500 μl of ethanol was performed. Further, the conditions for centrifuging without adding anything to the column before elution were room temperature, 20,000 xg for 5 minutes, and elution was performed with 30 μl of RNase Free Water.

(2.4. DNA extraction from organs and blood)
DNA extraction from the organ was performed using Quick Gene DNA tissue kit S (manufactured by KURABO) in the same manner as in the above section (1.2. PCR positive control).
DNA extraction from heparinized blood was performed using QIAamp (registered trademark) DNA Blood Mini Kit (manufactured by QIAGEN) according to the protocol attached to the kit.

(2.5. PCR)
For detection of RNA viruses, cDNA was prepared in the same manner as in the above section (1.2. PCR positive control). In addition, cDNA was also produced using Oligo dT ₂₀ (manufactured by Thermo Fisher Scientific) as a primer for reverse transcription.
cDNA reverse transcribed using these two types of primers was used to detect RNA viruses, and DNA was used to detect DNA viruses. Detection)
Note that for the Infected sample, a mixture of equal amounts of cDNA or DNA from each organ was used as a template.

<3. Results＞
(3.1. Detection of pathogens by PCR)
It was confirmed that conditions 1 to 3 shown above (1.3. Flow of establishing a detection system) were satisfied for 63 types of viruses and 1 type of protozoa shown in Tables 1-1 and 1-2. FIG. 1 shows the results of PCR product electrophoresis of 15 types of viruses to be detected by standard PCR and nested PCR.

FIG. 1(A) is a diagram showing electrophoresis results of PCR products as detection results by standard PCR. P in the figure indicates a positive control, and the positive control gene was mixed with cDNA derived from pig pancreas, which had been confirmed in advance that the target gene would not be amplified. N indicates a negative control, and the same cDNA as above was used. 100bp ladder marker (M) from the left end, followed by 2 wells each containing Getavirus, Sapovirus (type 3), (Type 7A), (Type 7B), (Type 7C), Porcine varicella virus, Porcine endogenous retrovirus ( PERV) (gag), (pol), (envA), (envB), (envC), swine epidemic diarrhea virus. The respective target sizes are 380bp, 330bp, 330bp, 330bp, 330bp, 350bp, 337bp, 810bp, 359bp, 263bp, 281bp, 377bp.
FIG. 1(B) is a diagram showing the electrophoresis result of the 2nd PCR product as a detection result by nested PCR. P and N in the figure were the same as in FIG. 1(A). 100bp ladder marker (M) from the left end, followed by 2 wells each containing hepatitis E virus (ORF1), hepatitis E virus (ORF2), rabies virus (type 7), hemagglutinating encephalomyelitis virus, porcine enterovirus, and pig rota. These are the results for a virus (type A), mammalian reovirus. The respective target sizes are 287 bp, 145 bp, 259 bp, 472 bp, 306 bp, 121 bp, and 344 bp.

In addition, 48 viruses detected by real-time PCR (African fever virus, Apoi virus, bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, bovine viral diarrhea virus type 3, bovine infectious Rhinobronchitis virus, Aujeski's disease virus, rabies virus 4, rabies virus 5, rabies virus 6, foot-and-mouth disease virus, porcine respiratory coronavirus, sapovirus type 6, sapovirus type 8, sapovirus type 9, sapovirus type 10 , vesicular stomatitis virus (new jersey type), western equine encephalitis virus, ton fever virus, Nipah virus, encephalomyocarditis virus, norovirus type A, norovirus type B, porcine astrovirus type 1, porcine astrovirus type 3, porcine astrovirus Virus type 5, porcine adenovirus, swine influenza virus type H1N1, swine influenza virus type H3N2, porcine cytomegalovirus, porcine circovirus type 1, porcine circovirus type 2, swine vesicular disease virus, swine infectious gastroenteritis virus, swine Torquetenovirus type 1, porcine torquetenovirus type 2, porcine endogenous retrourus (pol gene), porcine parvovirus, porcine reproductive and respiratory syndrome virus, porcine poxvirus, porcine lymphotropic herpesvirus type 1, porcine lymphotropic herpesvirus Melting curves were obtained for sexual herpesvirus type 2, porcine burravirus, porcine rotavirus B American type, porcine rotavirus B Vietnam type, porcine rotavirus C, bornavirus, Toxoplasma gondii, menangle virus, etc.). Illustrations are omitted.
As a negative control, cDNA derived from pig pancreas, which had been previously confirmed not to amplify the target gene, was used, and as a positive control, a mixture of the cDNA and a positive control gene was used. Note that NFW was used as a negative control for the porcine endogenous retrovirus (pol gene).

＊：ｒｅａｌ－ｔｉｍｅ ＰＣＲは２連で反応を行ったが、限界となる希釈段階において２連の内１つのみで目的産物の増幅が確認できた。
(3.2. Detection limit copy number)
The detection limit copy number of PCR is shown in Table 4 below using one significant digit. The detection limit by standard PCR was measured using 7 types of viruses, including 200 copies/μl for swine epidemic diarrhea virus, 300 copies/μl for swine rotavirus (type A), Getavirus, sapovirus (types 7A and 7B), The Japanese encephalitis virus was 2,000 copies/μl, and the sapovirus (type 3, type 7C) was 20,000 copies/μl. Nested PCR measures six types of viruses, two of which are hepatitis E virus (ORF1) and mammalian reovirus, with a detection limit of 50 copies/μl or less, and the detection limit for the remaining three is 1,000 to 2,000 copies/μl. It was μl. On the other hand, in real-time PCR, the detection limit for infectious bovine rhinobronchitis virus is 4,000 copies/μl, which is an order of magnitude higher than other pathogens, and the detection limit for other pathogens is 20 to 700 copies/μl. Ta.
The PCR efficiency is 58% for torquetenovirus (type 1), 55% for tonkholera virus, 54% for norovirus (type A), and 56% for type B, which is lower than other pathogens. It was 63% to 87%.

*: Real-time PCR was performed in two series, but amplification of the target product was confirmed in only one of the two series at the critical dilution step.

Regarding the detection sensitivity of pathogens, nested PCR, in which the number of PCR cycles is about 25 more than standard PCR, had higher detection sensitivity. On the other hand, it was confirmed that real-time PCR has a detection sensitivity that is about one order of magnitude higher than that of the above-mentioned two PCR methods for most pathogens. Therefore, it is preferable to unify all detection methods to real-time PCR.

(3.3. Evaluation of PCR using clinical specimens)
Figure 2 is a diagram showing the pathogen detection results obtained by evaluating the PCR test system using clinical specimens. (A) shows the melting curve as the detection result of porcine lymphotropic herpesvirus; Sample (Inf.) and negative control (N) are shown.
(B) shows an electrophoresis photograph of PCR products as bacterial detection results. From the left: 100bp ladder marker (M), positive control gene (P), negative control using NFW (N), infected sample (Inf.) . The target size is 464 bp.
(C) shows a melting curve as a result of detection of porcine cytomegalovirus, showing an infected sample (Inf.) and a negative control (N) that were performed in duplicate.
(D) shows an electrophoresis photograph of the PCR product as the detection result of Getavirus, from the left: 100bp ladder marker (M), positive control gene (P), negative control using NFW (N), and Clean sample 1. Liver (Liv.), Kidney (Kid.). The target size is 330bp. Since it was difficult to confirm the band with 35 cycles shown in Table 4, the results shown here are the results of increasing the number of cycles to 40.

Porcine endogenous retrovirus was detected in all organs of the infected sample and two clean samples.
Moreover, as shown in FIG. 2, in the Infected sample, porcine lymphotropic herpesvirus, bacteria, and porcine cytomegalovirus were also detected.
Getavirus was detected in the liver and kidney of Clean Sample 1 only when Oligo dT ₂₀ primer was used as the reverse transcriptase (Figure 2), and when the PCR product was confirmed by direct sequencing, it showed homology with Getavirus. Ta.
In addition, non-specific bands were observed in the detection of hepatitis E virus (ORF2), rabies virus (type 7), and swine vesicular virus, so when the PCR products were confirmed by direct sequencing, they were found to be PCR artifacts. It was confirmed that this was not the pathogen.

For confirmation of porcine cytomegalovirus, Hamel, AL, L. Lin, C. Sachvie, E. Grudeski, and GP Nayer. 1999. PCR assay for detecting porcine cytomegalovirus. J. Clin. Microbiol. 37:3767-3768 Detection was also performed using the primers reported in .
Figure 3 is a diagram showing the results of cytomegalovirus PCR product electrophoresis using the primers reported above, from the left: a 100bp ladder marker (M), a positive control gene (P), and a negative control using NFW. (N), Infected sample (Inf.). The target size was 413 bp, and a band of the target size was confirmed.

(3.4. Discussion)
In clinical specimens, in addition to porcine endogenous retrovirus, porcine lymphotropic herpesvirus, bacteria, and porcine cytomegalovirus were detected in infected samples by the testing method of the present invention.
Porcine lymphotropic herpesvirus and porcine cytomegalovirus are widely prevalent in healthy pig herds and do not normally show symptoms, so the pigs used as infected samples in this study showed clinical symptoms such as diarrhea and abdominal distension. The pathogen that caused this can be identified as not being caused by the above-mentioned virus, but by the bacteria detected by the present invention.
Since both viruses are subclinical infections, they are not considered a particular problem in the normal pig farming industry, but porcine lymphotropic herpesviruses have been suggested to be involved in lymphoproliferative diseases after allogeneic transplantation in pigs. has been done. In addition, porcine cytomegalovirus causes reproductive failure in those who do not have immunity to this virus. A risk of mother-to-child transmission has been suggested, although the possibility is low for porcine lymphotropic herpesvirus, and mother-to-child transmission often occurs for porcine cytomegalovirus, with 65% of piglets born to sows experimentally infected during gestation. There are also reports that this virus was isolated from As described above, according to the testing method of the present invention, it is possible to detect not only bacterial infection but also multiple types of viral infections from clinical specimens.

Above <1. Using the PCR detection system established in Establishment of a PCR-Based Pathogen Detection System, various pathogen infections were detected in the Infected samples, whereas it was detected that pathogen infections were significantly less in the above-mentioned Clean samples. This makes it possible to provide a material for xenotransplantation that is less susceptible to pathogen infection.
In particular, according to the testing method of the present invention, by using common primers, all 25 types of pathogens included in the guidelines of the Ministry of Health, Labor and Welfare were covered.

Claims (16)

The method includes the step of genetically testing infection of pathogens including two or more types of bacteria and viruses or protozoa, and in the genetic testing of the two or more types of bacteria, using primers for common base sequences between at least two types of bacteria. A method of testing xenograft materials for pathogen infection that detects the presence or absence of bacteria without identifying the species of bacteria . The method according to claim 1, wherein the common base sequence is a sequence included in a conserved region of bacterial 16S rRNA. The method according to claim 2, wherein the conserved region is a conserved region that is at least common to 16S rRNA of each of Leptospira, Mycoplasma, Campylobacter, Yersinia, E. coli, and Salmonella. 3. The method according to claim 2, wherein the conserved region is a region containing only V3 and V4 variable regions as variable regions, or a plurality of conserved regions sandwiching only V3 and V4 variable regions as variable regions. 2. The method of claim 1, wherein genetic testing for infection by a virus or protozoa comprises identifying the species of the virus or protozoa involved in the infection using a primer that selectively binds to nucleic acid of the virus or protozoa. . 3. The method according to claim 2, wherein the storage area is an area including V3 and V4 areas. The method according to any one of claims 1 to 6 , wherein one pair of primers is used in the genetic testing of the two or more types of bacteria. The genetic testing for infection by two or more types of bacteria and the genetic testing for infection by viruses or protozoa are performed by PCR under the same thermal cycle conditions, and the difference in melting temperature of all primers used is 15°C or less. The method according to any one of claims 1 to 7 , wherein: The method according to any one of claims 1 to 8 , wherein the pathogen infection is an infection that causes a zoonotic disease. The step of genetically testing for infection by viruses is a step for testing for infection by five or more types of viruses by PCR using a primer pair that selectively binds to the nucleic acid of each virus, or Any one of claims 1 to 9 , wherein the genetic testing step is a step of testing for infection by two or more types of protozoa by a PCR method using a primer pair that selectively binds to the nucleic acid of each protozoa. The method described in section. The method according to any one of claims 1 to 10 , wherein the xenograft material is a porcine-derived xenograft material. The virus includes porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus type H1N1, porcine lymphotropic herpesvirus, porcine cytomegalovirus, and hepatitis E virus, or the protozoa includes Toxoplasma gondii. , the method according to any one of claims 1 to 11 . A kit for testing pathogen infection of xenograft material, comprising a primer for a common base sequence between at least two types of bacteria and a primer that selectively binds to the nucleic acid of at least one type of virus or protozoa. , the kit for detecting the presence or absence of bacteria without specifying the species of the bacteria in the genetic testing of the at least two types of bacteria. A test kit comprising a primer for a common base sequence between at least two types of bacteria and a primer that selectively binds to the nucleic acid of at least one type of virus or protozoa,
The common base sequence is a sequence included in a conserved region of bacterial 16S rRNA,
The conserved region is a conserved region that is common to at least the 16S rRNA of Leptospira, Mycoplasma, Campylobacter, Yersinia, Escherichia coli, and Salmonella, and the genes of the at least two types of bacteria The test kit described above is for detecting the presence or absence of bacteria without specifying the species of bacteria . A test kit comprising a primer for a common base sequence between at least two types of bacteria and a primer that selectively binds to the nucleic acid of at least one type of virus or protozoa,
The common base sequence is a sequence included in a conserved region of bacterial 16S rRNA,
The conserved region is a region containing only V3 and V4 variable regions as variable regions, or a plurality of conserved regions sandwiching only V3 and V4 variable regions as variable regions, and for genetic testing of at least two types of bacteria, bacteria The test kit for detecting the presence or absence of bacteria without specifying the species . A method for producing a material product for xenotransplantation evaluated for pathogen infection, comprising the step of implementing the method according to any one of claims 1 to 12 .