JP6451002B2 - Rapid inspection method and system for Salmonella in feed - Google Patents

Description

  The present invention relates to a rapid inspection method and system for Salmonella in feed. More specifically, when examining Salmonella in the feed, the feed is first cultured to obtain a Salmonella enrichment culture, and then the nucleic acid is extracted from the enrichment culture using the polymer porous membrane filter. The present invention relates to a rapid test method and system for Salmonella in feed, which is extracted and detects the extracted nucleic acid by a PCR method, which is more sensitive than conventional methods and has a shorter test time.

  Contamination by bacteria belonging to the genus Salmonella (hereinafter referred to as “Salmonella”) causes serious problems in the feed industry. That is, Salmonella contamination in feed becomes a source of infection for livestock and the like, leading to contamination of food produced based on livestock. As a result, it cannot be denied that the food may cause food poisoning in humans. Therefore, in order to prevent feed contaminated with pathogenic microorganisms such as Salmonella from being manufactured, used and sold, feed provided to livestock is strictly regulated by laws on ensuring the safety of feed and improving quality. Has been. For example, in 1998, the Ministry of Agriculture, Forestry and Fisheries notified the “Guidelines for Salmonella Countermeasures for Feed Production” (revised in 2007), and thereafter the management system at the feed factory was strengthened. Therefore, although the salmonella positive rate of feed has been decreasing in recent years, it still remains at a positive rate of around 2%, which is not a reliable situation.

  In addition, the above “Guidelines” require the feed factory to conduct periodic inspections of feed ingredients, products, and Salmonella during the manufacturing process. In 2008, the “Feed Analysis Standard” was notified, Stipulates a culture method (so-called official culture method) for examining Salmonella. However, this official culture method requires 5 days to understand the results, so it lacks practicality in feed factories where quickness and simplicity are required, and instead of the official culture method, a quick and simple test kit is available. Inspection by law is also permitted in this guideline. Therefore, until now, the rapid inspection method of Salmonella in feed by the simple inspection kit method has been energetically studied, but it still takes 2-3 days to complete the inspection, and there is a more rapid and simple method. It was sought after.

  Therefore, a simple rapid inspection method using the PCR principle such as Qualibax (registered trademark) system has appeared (Non-Patent Document 1). In Non-Patent Document 1, for Salmonella in feed, the official culture method is compared with the inspection method (hereinafter referred to as “BAX method”) using an automatic measurement device Qualibax (registered trademark) system, and quality control in the feed factory is performed. It has been confirmed that the BAX method is as useful as the official culture method as the test method used for the test. However, even when such a BAX method is used, a minimum of 24 hours is required from the preparation of the sample to the determination, and a more rapid and simple method has been demanded.

  On the other hand, in recent years, there has been a remarkable progress in the method of recovering nucleic acids, removing impurities in a sample containing nucleic acids by a desired method, adsorbing and recovering nucleic acids on a solid phase carrier, washing the carrier, Numerous techniques for extracting and purifying nucleic acid by means of adsorbing and immobilizing nucleic acid on a so-called solid phase, such as recovering nucleic acid from a liquid phase have been proposed. For example, Non-Patent Document 2 proposes a method for extracting nucleic acid from a biological sample quickly and easily by adsorbing nucleic acid to a polymer porous membrane filter and then desorbing it. A rapid and simple nucleic acid extraction system using this polymer porous membrane is called Quick Gene (registered trademark). Until now, such a method is used to extract nucleic acid from whole blood or animal tissue. Although it has a certain track record in the medical industry, it has no track record in the feed industry, and no attempt has been made as to whether or not nucleic acid can be extracted from the enrichment culture solution of feed containing Salmonella.

Tetsuo Chihara et al., “Examination of rapid detection method of Salmonella in feed by Qualibax TM system”, Journal of Japanese Society of Food Microbiology, 2008, Vol. 25, No. 3, p. 109-119 Yoshihiko Makino et al., “Quickgene-800: Development of a rapid and simple nucleic acid extraction system using a porous polymer membrane”, FUJIFILM RESEARCH & DEVELOPMENT, 2006, No. 51, p. 39-47

  An object of the present invention is to provide a rapid inspection method and system for Salmonella in feed, which is more sensitive than the prior art and has a shorter inspection time.

  As a result of earnest research on the rapid inspection method for Salmonella in the feed, the present inventors first cultivated the feed to obtain a Salmonella enrichment culture, and then obtained a polymer porous medium from the enrichment culture obtained. It was found that by extracting a nucleic acid using a quality membrane filter and detecting the extracted nucleic acid by a PCR method, Salmonella in the feed can be detected quickly and easily, and the present invention has been completed. Surprisingly, it has been found that when this inspection method is employed, not only the sample preparation work is accelerated, but also salmonella in the feed can be detected with extremely high sensitivity. Furthermore, when extracting a nucleic acid, when the washing | cleaning process was abbreviate | omitted, it discovered that an inspection with few errors and high sensitivity could be performed. In addition, as a result of establishing such a highly sensitive test method, it was found that the conventional pre-enrichment culture time can be greatly shortened.

That is, according to one aspect of the present invention, a method for quickly testing Salmonella in a feed,
(A) culturing the feed in a medium to obtain a Salmonella enrichment culture,
(B) a step of extracting Salmonella nucleic acid from the obtained enrichment culture using a polymer porous membrane filter;
(C) A method comprising a step of detecting the extracted nucleic acid by a PCR method can be provided.
According to the preferable aspect of this invention, the said culture medium can provide the rapid test | inspection method of the Salmonella in feed which is a MP culture medium.
According to a preferred embodiment of the present invention, it is possible to provide a rapid test method for Salmonella in feed, wherein the PCR method is real-time PCR.
According to a preferred aspect of the present invention, it is possible to provide a rapid inspection method for Salmonella in feed that does not include a washing step in the extraction of the nucleic acid.
According to a preferred embodiment of the present invention, it is possible to provide a rapid inspection method for Salmonella in feed, wherein the extraction of the nucleic acid is performed with Quickgene (registered trademark).
According to a preferred aspect of the present invention, it is possible to provide a rapid inspection method for Salmonella in a feed in which the detection of the nucleic acid is performed by Qualibax (registered trademark).
According to the preferable aspect of this invention, the detection method of the said Salmonella can be provided within 9 to 11 hours, The rapid test | inspection method of the Salmonella in feed can be provided.
Moreover, according to one aspect of the present invention, a system for quickly inspecting Salmonella in feed,
(A) a medium for enriching Salmonella in the feed,
(B) a nucleic acid extraction device using a polymer porous membrane filter,
(C) It is possible to provide a system including a nucleic acid detection device using a PCR method.
According to a preferred aspect of the present invention, it is possible to provide a system for rapidly examining Salmonella in feed, wherein the medium is an MP medium.
According to a preferred aspect of the present invention, it is possible to provide a system for rapidly examining Salmonella in feed, wherein the PCR method is real-time PCR.
According to a preferred aspect of the present invention, it is possible to provide a system for rapidly examining Salmonella in a feed that does not include a washing step in the extraction of the nucleic acid.
According to a preferred aspect of the present invention, it is possible to provide a system for rapidly examining Salmonella in feed, wherein the nucleic acid extraction apparatus is Quick Gene (registered trademark).
According to a preferred aspect of the present invention, it is possible to provide a system for rapidly examining Salmonella in feed, wherein the nucleic acid detection device is Qualibax (registered trademark).
According to a preferred aspect of the present invention, it is possible to provide a system for quickly inspecting Salmonella in feed, in which the inspection of Salmonella ends within 9 to 11 hours.

According to the present invention, after culturing the feed to obtain an enrichment culture solution of Salmonella, nucleic acid is extracted from the obtained enrichment culture solution using a polymer porous membrane filter, and the extracted nucleic acid is subjected to PCR. By detecting by the method, it is possible to provide a rapid inspection method and system for Salmonella in feed, which is more sensitive than conventional methods and has a shorter inspection time.
In addition, according to the present invention, in order to enable early detection of process contamination in a feed factory and quick shipment of a product whose safety has been confirmed, for example, not only can the expansion of damage be prevented, but also inventory storage fees can be prevented. Such a salmonella inspection method and system can be provided to the feed industry that is sufficiently profitable even when the initial cost of the apparatus and the cost of analysis consumables are taken into consideration.

FIG. 1 is a diagram showing a procedure for selecting a pre-growth medium. FIG. 2 is a diagram comparing Salmonella growth in each pre-growth medium. FIG. 3 is a diagram comparing the sensitivity of the normal method and the new inspection method using an artificially contaminated oil bottle and a naturally contaminated sample. FIG. 4 is a diagram comparing the sensitivity of a normal method and a new test method using a sample obtained by serially diluting a pure Salmonella culture solution. FIG. 5 is a diagram comparing the flow of the normal method and the new inspection method. FIG. 6 is an analysis of the cause of error occurrence (cleaning process). FIG. 7 is a diagram comparing the sensitivity when an artificially contaminated oil bottle is measured by a new inspection method that does not include a cleaning process with that of a normal method. FIG. 8 is a diagram comparing the sensitivity when a naturally contaminated sample is measured by a new inspection method that does not include a cleaning step with that of a normal method. FIG. 9 is a diagram in which shortening of pre-enrichment culture time in the case where artificially contaminated oil cake is measured by a new inspection method is examined. FIG. 10 is a diagram in which shortening of the pre-enrichment culture time in the case of measuring a naturally contaminated sample by a new inspection method is examined.

Hereinafter, the rapid inspection method and system for Salmonella in the feed of the present invention will be described in order.
In the present invention, “feed” means all poultry and the like that are taken orally for nutritional purposes. Specifically, when classified in terms of nutrient content, all of roughage, concentrated feed, inorganic feed, and special feed In addition, when classified from the aspect of official standards, all of mixed feed, mixed feed, and simple feed are included. Moreover, when classifying from the aspect of a feeding method, it includes all feeds that are fed directly, feeds that are mixed with other feeds, or feeds that are added to drinking water to replenish nutrients.

  In the present invention, “Salmonella” refers to a bacterium belonging to the genus Salmonella, which is a genus of the Enterobacteriaceae family of Gram-negative facultative anaerobes, and is mainly enteric bacteria that inhabit the digestive tract of humans and animals. It is a kind of. Some of the bacteria belonging to the genus Salmonella can infect humans and animals orally and cause food poisoning. Food poisoning caused by Salmonella is a common infectious disease.

In the present invention, “medium” includes any non-selective and / or mildly selective general bacterial growth medium known to those skilled in the art. For example, MP media, BPW, EEM bouillon, BLB media or similar types of media are included. The medium can be either a non-selective medium and / or a mildly selective medium that allows rapid recovery and growth of potentially damaged Salmonella, and further allows sufficient Salmonella growth, As a result, a medium is included that can be prepared to include at least one viable Salmonella if Salmonella is present in the initial sample.
Examples of suitable non-selective growth media include MP media (MP Media, MP), buffered peptone water (BPW), EEM broth (EEM), BTB lactose broth (Lactose Broth with BTB, BLB) and other non-selective and / or mildly selective media known to those skilled in the art. The above culture media are available from DuPont Co., Ltd. (MP media), Kanto Chemical Co., Inc. (BPW), and Merck (EEM media, LB media). Among these, a preferable medium used in the present invention is MP medium. In the present invention, it is preferable to use an MP medium because the culture time is shortened. That is, 18 hours are required when BPW medium, EEM medium, and LB medium are used, but can be shortened to 14 hours when MP medium is used.
As a medium component for enrichment, those suitable for growth of Salmonella are selected. For example, nutrient components such as meat extract, peptone and yeast extract, saccharides such as lactose and glucose, and media components containing a selective agent and antibiotics for suppressing the growth of bacteria other than Salmonella are used. As the saccharide, it is preferable to use lactose that is particularly assimilated by the coliform group that is highly likely to be present in the sample, in order to improve selectivity for Salmonella. As selective agents, malachite green, magnesium chloride, alkyl sulfates such as sodium lauryl sulfate, bile salts and the like are preferable, and addition of novobiocin effective against Proteus is effective as antibiotics. Novobiocin or its sodium salt can be added at a concentration of 1-50 μg / ml. Preferably the concentration of novobiocin is 20-40 μg / ml, with a more preferred concentration being about 25 μg / ml. Also, other antibiotics (eg vancomycin, penicillin, ampicillin and amikacin) can be added to the medium at appropriate concentrations.

  In the present invention, the “nucleic acid” means an oligonucleotide or polynucleotide, and the oligonucleotide or polynucleotide may be modified or may contain a modified base. Oligonucleotides are single-stranded polymers of nucleotides containing 2-60 nucleotides. A polynucleotide is a polymer of nucleotides comprising two or more nucleotides. The polynucleotide comprises a double stranded DNA, single stranded RNA, double stranded RNA or RNA / DNA heteroduplex comprising a oligonucleotide whose second strand is annealed with an oligonucleotide of the reverse complement sequence of the first oligonucleotide. It can be a single stranded nucleic acid polymer. Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, rRNA, tRNA, and segmented nucleic acid.

  In the present invention, “nucleic acid” may include a label, and the label may mean a nucleotide, a nucleotide polymer, or any chemical moiety bound to a nucleic acid binding agent, It can be a bond or a non-covalent bond. Desirably, the label may be the nucleotide or nucleotide polymer that is detectable and can be detected by the experimenter of the present invention. Detectable labels include luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescent quenching agents, tonal molecules, radioisotopes and the like. The detectable label can be any useful linker molecule (eg, biotin, avidin, etc.), heavy metals, enzymes (eg, alkaline phosphatase, peroxidase and luciferase, etc.), electron donor / acceptor, acridium ester, dye and calorie. Includes measurement substrate. Those skilled in the art can also readily recognize useful detectable labels not mentioned above, which are also used in the practice of the present invention.

  The target Salmonella nucleic acid sequence for nucleic acid amplification can be selected from Salmonella nucleic acid sequences known in the art. This target nucleic acid sequence includes, but is not limited to, subspecies: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI) is not. Also included are, but are not limited to, nucleic acid sequences derived from Salmonella enterica species and Salmonella vongori species. For example, the serogroup and serotype of the subspecies Salmonella enterica can be confirmed in US Pat. No. 7,659,381.

Exemplary Salmonella nucleic acid sequences that are targeted for amplification in the present invention are disclosed in the following references, which are hereby incorporated by reference:
LiuWQ et al. , “Salmonella paratyphi C: genetic divergence from Salmonella choleraesuis and pathogenic convergence with Salmonella typhi”, PLoS One, 2009; 4 (2): e4510; Thomson NR et al. , “Comparative genome analysis of Salmonella enteritidis PT4 and Salmonella gallinarum 287/91 provides insights into evolutionary and host adaptation pathways,” Genome Res, 2008 Oct; 18 (10): 1624-37; Encheva V et al. "Proteome analysis of serovars typhimurium and Pullorum of Salmonella enterica subspecies I", BMC Microbiol, 2005 Jul 18; 5:42; McClell and M et al. "Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid", Nat Genet, 2004 Dec; 36 (12): 1268-74; Chiu CH et al. , “Salmonella enterica serotype Choleraesuis: epidemiology, pathogenesis, clinical disease, and treatment,” Clin Microbiol Rev, 2004 Apr; 17 (2): 311-22; Deng W et al. "Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18," J Bacteriol, 2003 Apr; 185 (7): 2330-7; Parkhil lJ et al. , “Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi C T18”, Nature, 200 1 Oct 25; 413 (6858): 848-52; McClell and M et al. "Complete genome sequence of Salmonella enterica serovar typhimurium LT2," Nature, 2001 Oct 25; 413 (6858): 852-6.
Salmonella enterica subsp. enterica serovar typhimurium str. An exemplary nucleotide sequence of the complete genome of LT2 (4857432 bp) can be confirmed with GenBank Accession number: NC_003197.
Those skilled in the art can appropriately prepare probes and primers for detecting Salmonella in the present invention based on these nucleic acid sequences.

The target nucleic acid sequence on the Salmonella genome is PCR, reverse transcriptase PCR (RT-PCR), real-time PCR, real-time RT-PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), any other Can be detected by conventional techniques such as isothermal nucleic acid amplification, nucleic acid probes, and / or biosensors.
Here, real-time PCR combines nucleic acid amplification and fluorescence detection of the amplified nucleic acid. Briefly, when a standard PCR intended for amplification of a target nucleic acid is bound to the target nucleic acid, fluorescence is emitted. It is carried out in the presence of a probe that specifically generates a signal and is monitored by monitoring the fluorescence emission during the PCR cycle. The cycle in which the fluorescence emitted from the PCR is measured above the threshold (ie, above the background fluorescence level) is called the threshold cycle (Ct). Ct has been shown to be proportional to the common logarithm of the amount of target nucleic acid initially present in PCR. Thus, measurement of Ct allows measurement of the initial concentration of target nucleic acid in the sample.

  In the present invention, it is preferable to use real-time PCR or real-time RT-PCR from the viewpoint of detecting Salmonella efficiently and rapidly. Moreover, as a specific example suitably used in the present invention, there is a Qualibax (registered trademark) system. In the Qualibax (registered trademark) system, the double strand of the PCR-amplified target nucleic acid fragment binds to cyber green and emits fluorescence. However, as the temperature rises, the strand is denatured into a single strand and disappears. The melting curve profile obtained by plotting this optical signal in the range of 70 to 95 ° C. is automatically analyzed to determine positive or negative. Note that indeterminate is displayed, and determination may be suspended (error). In addition, the melting curve profile can be confirmed for each sample, and when Salmonella is positive, a control peak in the range of 78 to 80 ° C and a target peak at 85, 88, and 90 ° C are recognized, If it is negative, only the control peak is observed. From the difference, it can be determined whether the sample is positive or negative.

  In the present invention, a nucleic acid extraction method is used in which a Salmonella nucleic acid is adsorbed on a polymer porous membrane filter and recovered, and then the nucleic acid is recovered from the filter. In this method, since the entire amount of nucleic acid can be recovered from a sample containing only a small amount of nucleic acid, there is an advantage that even a sample having a low nucleic acid content can be recovered by concentrating the nucleic acid on a polymer porous membrane filter. For this reason, a small amount of nucleic acid can be subjected to an amplification reaction without loss, and a highly sensitive measurement is possible. Therefore, the conventional secondary culture of Salmonella can be omitted, and Salmonella lysis and nucleic acid extraction can be performed at the same time, so that the steps necessary for the inspection of Salmonella in the feed can be shortened (FIG. 5). See).

In carrying out the present invention, there is no particular limitation as long as it is a polymer porous membrane filter capable of adsorbing and recovering nucleic acids. For example, a surface saponified product of acetylcellulose is preferable. As acetyl cellulose, any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose may be used, and triacetyl cellulose is particularly preferable.
As a suitable specific example of the polymer porous membrane filter, a Quick Gene (registered trademark) system is exemplified. This Quick Gene (registered trademark) system was developed as an automated system for extracting nucleic acids from biological samples quickly and easily, and consists of a polymer porous membrane filter and treatment solution kit, and a dedicated automated machine. It is. The membrane of this system is very thin (80 μm) compared to glass fiber (1 mm), and has an advantage in that a nucleic acid with less impurities can be obtained. In addition, the pore diameter of the membrane is high in uniformity, suitable for filtration by air pressurization, and can be downsized. Increasing the hydrophilicity of the filter surface increases the yield of nucleic acid, but the polymer porous membrane filter is made of organic polymer, not glass fiber, so the surface can be easily modified with various functional groups. It is possible to control the adsorption and desorption of nucleic acids to the filter. In this system, it is possible to adsorb and desorb nucleic acids on the filter surface by appropriately controlling the surface characteristics of the filter and the polarity of the lysate, washing solution, and recovery solution containing the nucleic acid. For example, when the polarity of the lysate is lowered by adding an organic solvent such as ethanol, the nucleic acid is adsorbed on the filter. By washing the filter with a liquid having a low polarity before release, components other than the nucleic acid remaining on the filter in a state where the target nucleic acid is adsorbed can be washed. Finally, the nucleic acid is eluted and collected with a highly polar solution. Through such a process, a nucleic acid sample with high purity can be obtained relatively easily. As will be described later, it is preferable not to include a washing step for the adsorption and recovery of nucleic acids.

  EXAMPLES Next, although an Example and a comparative example are given and this invention is demonstrated in more detail, this invention is not restrict | limited to these at all.

[Example 1] Selection of pre-enrichment medium In order to select an optimum medium as the pre-enrichment medium, the experiment shown in Fig. 1 was conducted. That is, the oil cake and Salmonella microbial solution were mixed to obtain an oil cake of 10 ⁰ cfu / 25 g (cfu represents a colony forming unit). In addition, the tested strains are two strains derived from the production environment (groups O1, 3, 19 and O7). Put 25g each into 4 stomacher bags, pour BPW, EEM medium, LB medium and MP medium, respectively, cultivate BPW, EEM medium and LB medium at 35 ° C for 18 hours, and MP medium at 42 ° C. Cultured for 14 hours.
The result is shown in FIG. As is clear from this figure, when MP medium is used, active growth is obtained from the initial stage of culture time of 1 to 5 hours, and relatively high enrichment efficiency is obtained over culture time of 5 to 14 hours. It was. On the other hand, when BPW, EEM medium, and LB medium were used, there was almost no increase in bacteria at the initial stage of culture time of 1 to 5 hours, and only a low increase efficiency was obtained compared with MP medium at culture time of 5 to 14 hours. I couldn't. Therefore, this experiment revealed that MP medium is particularly preferable as a medium for enriching Salmonella. Therefore, it was decided to use MP medium in the following experiment.

The method of the present invention is hereinafter referred to as a “new inspection method”, and the method that has been performed so far is referred to as a “normal method”.
[Example 2] Salmonella inspection method (new inspection method)
(1) Preparation of material 19 samples of 10 ⁰ cfu / 25 g oil candy were obtained as samples for the experiment using artificially contaminated oil candy. In addition, 8 samples of 10 ¹ cfu / 25 g oil candy were obtained as samples. In addition, the tested strain is one strain derived from the production environment (groups O1, 3, 19).
For the experiment using a natural contamination sample, 32 samples of dust derived from the manufacturing environment were prepared as samples, and 6 samples of work floors (wiping off) were prepared.
The sample was subjected to pre-enrichment culture in MP medium for 14 hours to obtain an enrichment culture solution of Salmonella.
(2) Preparation of lysis sample 200 μl of the enrichment culture solution was put in a 2 ml PCR tube, and 180 μl of MDT (tissue lysis buffer) and 20 μl of EDT (proteinase K) were added thereto. This was vortexed at 2500 rpm for 15 seconds and allowed to stand at room temperature for 2 minutes. Further, 180 μl of LDT (lysis buffer) was added and vortexed at 2500 rpm for 15 seconds. Next, this was heated at 70 ° C. for 10 minutes, 240 μl of ethanol was added, and vortexed at 2500 rpm for 15 seconds. By the above operation, a Salmonella lysate solution was obtained.
(3) Purification of DNA Quick DNA (registered trademark) system (manufactured by Kurabo Industries) was used for DNA purification. The lysate obtained as described above was transferred to a dedicated cartridge (including a polymer porous membrane filter) and set in a cartridge holder. Next, the dedicated cartridge was pressurized to discard unnecessary solutions, 750 μl of WDT (washing buffer) was added, and the pressure was discarded to discard the washings. This washing operation was repeated three times to complete the washing step, and the DNA was purified.
(4) Preparation of detection sample After finishing the purification in (3) above, the setting position of the tube holder of the pump was switched from disposal to extraction. 200 μl of qualibux (registered trademark) lysis solution was added, and the sample containing nucleic acid was recovered by pressurization. Of these, 30 μl is a sample of the Qualibax (registered trademark) system.
(5) Detection of DNA For detection of DNA, Qualibax (registered trademark) system (manufactured by DuPont) was used. The 30 μl sample was mixed with a tablet reagent and set in a Qualibax (registered trademark) system apparatus. The test result was judged after about 1 hour. This Qualibax (registered trademark) system was performed by real-time PCR, and the determination of the test result was displayed by a positive determination icon or a negative determination icon. The result is shown in FIG. FIG. 3 shows only the number of positive determinations.
The tablet reagent contains reaction materials such as polymerase, primer, probe and fluorescent reagent necessary for PCR. This tablet also contains an internal positive control, and it is not necessary to prepare a positive control or create a calibration curve. As described above, the Qualibax (registered trademark) system does not require a reagent preparation step, which is one of the most complicated steps in PCR, and thus is particularly preferable in the implementation of the present invention requiring rapidity.

[Comparative example] Salmonella inspection method (normal method)
(1) Preparation of material A material was prepared in the same manner as in (1) of Example 1 to obtain a Salmonella enrichment culture solution.
(2) Preparation of Bacteria Sample The enrichment culture solution was further cultured in a brain heart infusion (BHI) medium at 37 ° C. for 3 hours. On the other hand, 150 μl of protease was added to 12 ml of lysis buffer to prepare a lysis reagent. Dispense 200 μl of this lysis reagent into a PCR tube, add 5 μl of the above enrichment culture solution, heat at 37 ° C. for 20 minutes, further heat at 95 ° C. for 10 minutes, rapidly cool the tube with a cooling block, A lysate solution was obtained.
(3) Preparation of detection sample The purification of the DNA of Example 1 (3) was omitted, and a sample was prepared. To the Salmonella lysate solution, 50 μl of a lysis solution for Qualibax (registered trademark) was added to prepare a sample for the Qualibax (registered trademark) system.
(4) Detection of DNA As in (5) of Example 1 above, DNA was detected using the Qualibax (registered trademark) system (manufactured by DuPont). The results of this test are shown in FIG. FIG. 3 shows only the number of positive determinations.

As shown in FIG. 3, comparing the normal method and the new test method (QuickGene method), it was found that the new test method has higher detection sensitivity of Salmonella than the normal method. That is, in 10 samples of artificially contaminated oil bottles of 10 ⁰ cfu / 25 g, only 7 samples can be detected by the normal method, but 9 samples are detected by the new test method. Thus, it was suggested that the ability to detect Salmonella from a sample containing Salmonella at a low concentration is high in the new test method. Similarly, in the natural contamination sample 38 samples, only 17 samples can be detected by the normal method, but 19 samples are detected by the new test method. This also suggests that the new test method has higher detection sensitivity than the normal method.

[Example 3] Sensitivity analysis in the normal method and the new test method In the above Example 2 and the comparative example, it was suggested that the new test method exhibits higher detection sensitivity than the normal method. The pure Salmonella culture solution obtained in Example 1 (cultured in MP medium for 14 hours, 10 ⁸ cfu / ml) was diluted stepwise to give a bacterial concentration of 10 ⁴ cfu / ml, 10 ³ cfu / ml, 10 ² Samples of cfu / ml, 10 ¹ cfu / ml or 10 ⁰ cfu / ml were obtained and used as a starting point. Both the normal method and the new test method were performed twice as in Example 2 and Comparative Example. Carried out. The result is shown in FIG.

As is clear from the results of FIG. 4, the new inspection method showed a sensitivity 10 to 100 times higher than that of the normal method. In other words, the new test method was able to detect even a low-concentration sample having a bacterial concentration of 10 ² cfu / ml or 10 ¹ cfu / ml, but the normal method was able to detect a high bacterial concentration of 10 ³ cfu / ml or higher. Only concentration samples could be detected. From this, it was confirmed that the new inspection method has higher detection sensitivity than the normal method.
However, as shown in FIG. 4, when the new inspection method was used, an error occurred in a low concentration sample of 10 ¹ cfu / ml or 10 ⁰ cfu / ml. Therefore, the cause of the error was examined next.

[Example 4] Examination of cause of error As a result of intensive investigation of the process of the new test method, it was found that there was a cause in the washing process of washing out things other than DNA. Therefore, whether or not an error occurs in a low concentration sample having a bacterial concentration of 10 ¹ cfu / ml or 10 ⁰ cfu / ml by reducing the washing step (3) of Example 2 once in order from 3 times. It was confirmed. The result is shown in FIG.

  As shown in FIG. 6, an error occurred when the cleaning process was performed three or two times. On the other hand, no error occurred when the washing process was performed once or zero. From this, it was found that no error occurs when the number of times of washing is small, and that an error occurs when a cleaning reagent is mixed. If the cleaning process is not carried out, the risk of error occurrence is reduced, and the labor of the cleaning work is reduced, which is considered to be convenient for increasing the speed of inspection. Therefore, next, an experiment was conducted to determine how the detection sensitivity would be when the cleaning process was not performed.

[Example 5] Inspection method without performing the cleaning step An experiment was conducted to determine whether or not the new inspection method without the cleaning step exhibits higher sensitivity than the normal method.
(1) Experiment using artificially contaminated oil cake (10 ⁰ cfu / 25 g) In the same manner as in (2) of Example 2, samples that were artificially contaminated with 10 ⁰ cfu / rapeseed meal 25 g were used as a sample. The new inspection method was carried out in the same manner as in Example 2 and the comparative example. Ten samples were prepared. The result is shown in FIG.
(2) Experiments using in-process reservoirs (natural contaminants) In the same manner as (2) of Example 2, natural contamination samples were prepared, and the current method and new inspection method were made the same as in Example 2 and Comparative Example. Carried out. 18 samples were prepared. The result is shown in FIG.

As is apparent from FIGS. 6 and 7, the new inspection method without the washing step showed higher detection sensitivity than the normal method. That is, in an experiment using an artificially contaminated oil bottle, specimen # 10 was determined to be negative in the normal method, but positive in the new test method. Here, according to the official culture method, sample 10 was positive, and thus, in the normal method, one sample was missed.
In an experiment using natural contaminants, Sample No. 6 was judged negative by the conventional method but positive by the new test method. Here, according to the official culture method, since sample 6 was positive, one sample was missed in the normal method.
From the above results, it was found that the new inspection method that does not perform the cleaning step has higher sensitivity than the normal method and can make an accurate determination. It has also been found that sufficient sensitivity can be obtained even if the washing step is omitted. Therefore, the present invention preferably does not include a washing step in nucleic acid extraction.

[Example 6] Shortening of pre-culture time In Examples 1 to 5 so far, it was found that the new test method had good sensitivity. An experiment was conducted to see if the test results could be determined.
(1) Experiment using artificially-contaminated oil cake (10 ⁰ cfu / 25 g) In the same manner as in (1) of Example 2, samples 1 to 7 were artificially contaminated with 10 ⁰ cfu / 25 g. And the new inspection method was implemented like Example 2 and a comparative example. For each sample, the pre-enrichment culture time was 6 hours, 7 hours, 8 hours, and 9 hours. The result is shown in FIG.
(2) Experiments using in-process reservoirs (natural contaminants) In the same manner as (2) of Example 2, natural contamination samples were prepared, and the current method and new inspection method were made the same as in Example 2 and Comparative Example. Carried out. For each sample, the pre-enrichment culture time was 6 hours, 7 hours, 8 hours, and 9 hours. The result is shown in FIG.

As is clear from FIG. 8, it was found that the pre-enrichment culture time can be shortened to 9 hours in the experiment using the artificially contaminated oil bottle. Further, as is clear from FIG. 9, it was found that the pre-enrichment culture time can be shortened to 7 hours in the experiment using the naturally contaminated sample.
Thus, since the new test method has higher sensitivity than the normal method, it has been found that the pre-enrichment culture time, which conventionally required 14 hours, can be shortened to 7-9 hours. If the pre-enrichment culture time can be shortened to 7 to 9 hours, nucleic acid extraction (about 1 hour) using the Quickgene (registered trademark) system and nucleic acid detection (about 1 hour) using the Qualibax (registered trademark) system can be performed. Even when combined, Salmonella in the feed could be inspected in a total of 9 to 11 hours, and it was found that the inspection results were revealed during the day. In a feed factory that requires quickness and simplicity, it is very useful to find out the test results during the day, and it is clear that the present invention can be an effective method at the production site.

Claims (5)

A method for testing salmonella in feed,
(A) culturing the feed in a medium to obtain a Salmonella enrichment culture,
(B) obtaining a lysate from the resulting enriched culture;
(C) extracting Salmonella nucleic acid from the obtained lysate using a polymer porous membrane filter of Quickgene (registered trademark) system ;
(D) a step of detecting the extracted nucleic acid by a PCR method, the step of extracting the nucleic acid, the step of adsorbing the nucleic acid from the lysate solution to the polymer porous membrane filter, and the step of adsorbing the nucleic acid A step of recovering the nucleic acid from the polymer porous membrane filter, wherein the nucleic acid is adsorbed between the step of adsorbing the nucleic acid and the step of recovering the nucleic acid. Does not include the step of washing,
The method.   The method according to claim 1, wherein the medium is an MP medium.   The method according to claim 1 or 2, wherein the PCR method is real-time PCR.   The method according to any one of claims 1 to 3, wherein the examination of Salmonella is performed within 9 to 11 hours.   The manufacturing method of feed containing the inspection method of any one of Claims 1 thru | or 4.