JP6728608B2 - Sample preparation method for nucleic acid analysis - Google Patents

Description

The present invention relates to a sample preparation method for nucleic acid analysis, and a method for analyzing nucleic acid using a sample obtained by the method.

Sepsis is a serious systemic infection, and detection and identification of the causative microorganism in blood is essential for definitive diagnosis. There are various bacteria such as Staphylococcus aureus that are the cause of sepsis. In particular, it is not uncommon for Staphylococcus aureus to have multidrug resistance called methicillin-resistant Staphylococcus aureus (MRSA), and in order to select the appropriate antibacterial drug and save the patient, It is clinically important to detect and identify quickly.

Since it is difficult to directly test the causative microorganisms in blood, generally, patient blood is added to, for example, a blood culture bottle and cultured to prepare a blood culture solution in which the causative microorganisms are grown, and this is separated and cultured. And used for genetic testing.

In the field of microbial testing, genetic testing has the advantage of being detected and identified very quickly. In the conventional genetic test using a blood culture solution as a sample, a method of extracting nucleic acid from the microbial cells in the blood culture solution and subjecting it to a genetic test, or collecting the microbial cells in the blood culture solution and lysing them before gene The method used for inspection has been mainly used. (Non-patent document 1, non-patent document 2)
These methods have the drawback that the speed of the test is lost due to the labor and time required for the nucleic acid extraction step or the lysis step.

As another example of the method, there is a method in which a blood sample is filtered, and nucleic acids are extracted from the filtered sample to be analyzed (Patent Document 1). Although only the blood sample is targeted in this document, it is possible that the same method may be used because blood is also contained in the blood culture solution. However, the method described in the document is the same as the above-mentioned method in that the sample still needs to be filtered and the nucleic acid is extracted, and the problem that the speed of the test may be lost due to the complicated work remains.

As another method, there is a method of diluting a blood culture solution with sterile distilled water, adding a lytic enzyme to the diluted solution, heating the solution, and lysing the solution by enzyme treatment (Patent Document 2). In this document, a Staphylococcus aureus-specific lytic enzyme called lysostaphin is used. The method does not require nucleic acid extraction, but requires an enzyme used for lysis and a warmer for heating. Further, it may be necessary to use different lytic enzymes depending on the type of bacteria, so it is considered difficult to carry out the method when the bacterial species in the culture solution to be analyzed is unknown.

Special table 2009-537167 Special table 2002-537824

Journal of Clinical Microbiology, September 2000, p. 3407-3412 The Journal of Molecular Diagnostics, March-April 2012, p. 120-129

The present invention is intended to solve the above problems associated with genetic testing.

As a result of intensive studies in view of the above problems, the present inventors have invented a method of preparing a precipitate from a blood culture solution by a very simple method and detecting a nucleic acid directly from the precipitate.
That is, the present invention has the following configurations.
[Item 1]
A method for preparing a sample for nucleic acid analysis, which comprises only the following steps (I) to (III).
(I) Step of preparing a suspension by adding a liquid containing no surfactant to a blood culture solution and mixing them (II) Centrifuging the suspension prepared in step (I) to separate a supernatant and a sediment Step (III) of removing the supernatant from the prepared solution, a step of adding a liquid containing no surfactant to the precipitate remaining in the step (II), and mixing the mixture to prepare a suspension [Item 2]
Item 2. The method according to Item 1, wherein after the step (III), a cycle of performing the following step (II′) and then performing the step (III) is inserted once or plural times.
(II′) A step of centrifuging the suspension prepared in the step (III) to remove the supernatant from the solution separated into the supernatant and the sediment [Item 3]
The method according to Item 1 or 2, further comprising the following step (VI).
(IV) A step of diluting the sediment suspension prepared in the step (III) 2 to 10000 times with a liquid containing no surfactant [Item 4].
Item 4. The method according to any one of Items 1 to 3, wherein the liquid containing no surfactant is any one or more selected from the group consisting of water, a buffer solution and an alkaline solution.
[Item 5]
Item 5. The method according to Item 4, wherein the liquid containing no surfactant is water.
[Item 6]
A method for analyzing nucleic acid, which comprises analyzing a nucleic acid contained in a sample for nucleic acid analysis prepared by the method according to any one of Items 1 to 5.

INDUSTRIAL APPLICABILITY The present invention prepares a sample for nucleic acid analysis from a blood culture solution using only a liquid containing no surfactant, neither purification of the culture liquid nor lysis with a liquid containing a surfactant or the like. It is characterized by doing. Conventionally, a step of further purifying nucleic acid from a blood culture solution or a step of lysing bacteria in a sample was required, but the present invention can prepare the sample without performing these steps.

Further, according to the present invention, the use of the sample enables rapid and simple detection of nucleic acid (for example, nucleic acid derived from a microorganism) from a blood culture solution. Furthermore, since the present invention does not require special equipment or reagents, it requires almost no new labor or equipment to carry out the invention. Further, in the present invention, since a surfactant which may cause a difference in quality, reagent contamination, spoilage, etc. is not used, stable measurement can be performed.

In addition, the nucleic acid analysis possible in the present invention is not limited to a specific gene or base sequence, and various genes derived from microorganisms cultured by blood culture can be analyzed.

FIG. 5 is a diagram showing the results of Example 1.

One aspect of the embodiment of the present invention is a method for preparing a sample for nucleic acid analysis, which comprises only the following steps (I) to (III).
(I) Step of preparing a suspension by adding a liquid containing no surfactant to a blood culture solution and mixing them (II) Centrifuging the suspension prepared in step (I) to separate a supernatant and a sediment Step (III) of removing the supernatant from the prepared solution, a step of adding a liquid containing no surfactant to the precipitate remaining in the step (II) and mixing them to prepare a suspension

In the above method, after step (III), a cycle of carrying out step (II′) below and then step (III) may be inserted once or plural times.
(II') Step of centrifuging the suspension prepared in step (III) to remove the supernatant from the solution separated into a supernatant and a sediment Here, the number of cycles to be inserted is not particularly limited, but preferably 1 It is 10 times, more preferably 1 to 5 times, further preferably 1 to 2 times, and further preferably 1 time.
The cycle is a step in which the precipitate obtained in step (II) is washed once or more times. By adding this washing step, the step of analyzing nucleic acid can be performed more accurately.

In the method, in step (I), the liquid volume ratio of the blood culture solution and the liquid containing no surfactant is not particularly limited as long as the liquid containing no surfactant is equal to or more than the liquid containing the blood culture. The ratio of the blood culture solution and the liquid containing no surfactant is preferably 1:1 to 1:10, more preferably 1:3 to 1:6.

In the above method, the following step (VI) may be further carried out after the step (III) (when all the cycles are completed when the cycle is inserted in the step).
(IV) A step of diluting the sediment suspension prepared in the step (III) by a factor of 2 to 10000 with a liquid containing no surfactant.

The dilution ratio at this time is not particularly limited as long as it is within the above range. The preferable lower limit is 3 times, more preferably 4 times, and further preferably 5 times. The preferable upper limit is 1000 times, more preferably 100 times.

Blood culture is inoculation of blood collected from a patient into a medium-containing bottle or the like and culture in order to identify pathogenic microorganisms of serious infectious diseases such as sepsis. The “blood culture solution” used in the present invention refers to, for example, a liquid portion in a blood culture bottle after blood culture.

The sample preparation method for nucleic acid analysis of the present invention does not include a step of purifying nucleic acid. The “step of purifying nucleic acid” refers to a method of actively separating nucleic acid and contaminants from a sample. Contaminants include components of blood culture bottles as well as biopolymers of the microorganisms themselves that have the nucleic acid of interest. Examples of the method include a method of adsorbing nucleic acid on silica to separate it from components other than the nucleic acid.

The term "surfactant-free liquid" used in the present invention refers to a liquid or solution containing no ionic or nonionic surfactant. The pH and other components are not particularly limited as long as the surfactant is not included. It is more preferably any one or more selected from the group consisting of water, a buffer solution and an alkaline solution, and more preferably water.

The "surfactant-free liquid" used in step (I), step (III) and step (IV) need not be the same in all steps. Different ones may be used in all steps, or the same one may be used in any two steps and another one may be used in another step. Moreover, you may use the same thing in all the processes.
Preferred is a combination of water and a buffer solution or water and an alkaline solution.

The “water” used in the present invention is not particularly limited as long as it is free of microorganisms. Examples thereof include distilled water, ion-exchanged water, membrane-permeated water, purified water such as ultrapure water or pure water that has been sterilized by an autoclave, and water for injection that is commercially available for medical use. Does not include tap water or well water that may be contaminated with microorganisms and contaminants.

The “buffer solution” used in the present invention refers to a solution having a buffering capacity. Specific examples thereof include Tris buffer, Good's buffer, and various buffers commonly used in the field of molecular biology, and Tris buffer is more preferable.
The pH of the buffer solution is preferably 7 or more. A more preferable lower limit of pH is 7.5. The preferable upper limit of the pH of the buffer solution is 9.
The concentration of the buffer solution is preferably 10 mM or more. The preferable upper limit of the buffer solution is 100 mM. A more preferable upper limit of pH is 20 mM.

The “alkaline solution” used in the present invention refers to a basic liquid having a pH of 8.0 or higher at 25° C. and having no buffering ability. There is no particular limitation as long as it satisfies this condition, but a basic liquid having a concentration of 50 mM or less is preferable, and a basic liquid having a concentration of 20 mM or less is more preferable. An example of such a liquid is an aqueous solution of an alkali metal hydroxide. Preferred is a potassium hydroxide solution or a sodium hydroxide solution.

One aspect of the embodiment of the present invention is a nucleic acid analysis method for analyzing a nucleic acid contained in a sample for nucleic acid analysis prepared by any of the methods described above.
The “method for analyzing nucleic acid” in the present invention is a method for detecting a nucleic acid or a method for identifying a microorganism by detecting a nucleic acid. A conventionally known method can be used as the method for detecting a nucleic acid and is not particularly limited, but preferably, two methods of (1) a step of amplifying a nucleic acid by a nucleic acid amplification reaction and (2) a step of detecting the amplification product are performed. It is a method including steps.

Examples of the nucleic acid to be analyzed in the present invention include DNA or RNA derived from microorganisms, but other nucleic acids that can be contained in a blood culture solution are also analyzed. For example, when a microorganism that has undergone gene recombination to synthesize an artificial nucleic acid such as PNA (Peptide Nucleic Acid) is cultured in blood culture, the present invention can be used to easily prepare an artificial nucleic acid such as PNA. It is also possible to detect.

The specific nucleic acid amplification method used in the nucleic acid amplification step is not particularly limited, and a known method can be appropriately used. For example, a PCR (Polymerase Chain Reaction) method, a NASBA (Nucleic acid sequence based amplification) method, a TMA (Transcription-mediated amplification) method, an SDA (Stranded Displacement) method, or the like is preferable. In each of these methods, the conditions of the amplification reaction are not particularly limited and can be carried out by a conventionally known method.

The specific nucleic acid detection method used in the step of detecting a nucleic acid amplification product is not particularly limited, and a known method can be appropriately used. For example, an agarose gel electrophoresis method, a sequencing method, a DNA probe method, a real-time PCR method and the like can be mentioned.

The microorganism to be detected by the method of the present invention or the microorganism from which the nucleic acid to be detected by the method of the present invention is derived is not particularly limited as long as it can be cultured in a blood culture bottle or the like.

In the method of the present invention, the blood culture method is not particularly limited. For example, a commercially available blood culture bottle may be used for culturing by a standard method.
The medium used for blood culture is not particularly limited, and for example, the medium contained in a commercially available blood culture bottle can be used as it is.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.

[Example 1: Direct detection of coagulase-negative staphylococci cultivated in a blood culture bottle]
(1) Preparation of sample 150 μL of a blood culture solution containing methicillin-resistant coagulase-negative staphylococcus was mixed with 800 μL of purified water, and the mixture was centrifuged at 3000 g for 3 minutes to remove the supernatant and leave only the precipitate. This precipitate was suspended in 500 μL of purified water (ion-exchanged water), and the suspension was used as a sample (R1). Separately, the precipitate was suspended in 500 μL of TE buffer (10 mM, pH 8.0), and the suspension was used as a sample (R2).
In addition, a blood culture solution containing methicillin-sensitive coagulase-negative staphylococcus was subjected to the same treatment as described above to prepare samples (S1, S2).

(2) Nucleic Acid Amplification The following reagents were added to the above-mentioned sample or purified water (used as a negative control), and the methicillin resistance gene (mecA gene) was detected by the PCR method. Nucleic acid amplification was performed under the following conditions. GeneAmp PCR System 9700 (manufactured by Applied Biosystems) was used for nucleic acid amplification.

(3) Detection of Nucleic Acid Amplification Product The nucleic acid amplification product was confirmed by electrophoresis using 1×TAE agarose gel. The electrophoresis was performed at 100 V for 30 minutes, the agarose gel after the electrophoresis was stained with ethidium bromide, and the amplified product was visualized and confirmed by irradiation with ultraviolet rays.

Reagents A 20 μL solution containing the following reagents was prepared.
0.2 μL of 10 μM nucleic acid primer for amplifying mecA gene (SEQ ID NO: 1)
Nucleic acid primer for amplifying mecA gene (SEQ ID NO: 2) 10 μM 0.2 μL
×10 buffer 2 μL
2 mM dNTP 2 μL
Blend Taq polymerase (manufactured by Toyobo) 0.5U

Sample or purified water 1 μL

Nucleic acid amplification conditions 94℃, 2 minutes (1 cycle above)
94°C for 1 minute 55°C for 30 seconds 72°C for 1 minute 30 seconds (37 cycles above)
72℃, 3 minutes and 30 seconds (1 cycle above)

Results FIG. 1 shows the results of electrophoresis. No amplification product of the mecA gene was detected in methicillin-sensitive coagulase-negative staphylococci. On the other hand, an amplification product of the mecA gene was detected in methicillin-resistant coagulase-negative staphylococci. No difference was found in the results whether the liquid suspending the precipitate was purified water or TE buffer. From the above, it was shown that by carrying out the present invention, a nucleic acid analysis sample can be easily prepared from a blood culture sample, and that the sample can be prepared using purified water or a buffer.

[Example 2: Detection of Staphylococcus aureus in blood culture by real-time PCR method]
(1) Preparation of sample 200 μL of a blood culture solution in which Staphylococcus aureus was cultured and 600 μL of purified water were mixed, and the mixture was centrifuged at 3000 g for 3 minutes to remove the supernatant and leave only the precipitate. Purified water (900 μL) was added to the precipitate and the mixture was centrifuged again, and the precipitate was suspended in 500 μL of purified water. This suspension was used as it was or diluted with purified water 10 times, 100 times, 1000 times, 10000 times, and 100000 times as a sample.

(2) Real-time PCR method The following reagents were added to the above samples, respectively, and the Staphylococcus aureus-specific nuclease gene (nuc gene) was detected using the real-time PCR method. Detection was confirmed by calculation of Ct value. Real-time PCR was performed under the following conditions. Rotor-Gene Q (manufactured by Qiagen) was used for real-time PCR.

Reagents A 20 μL solution containing the following reagents was prepared.
Nuc gene amplification nucleic acid primer (SEQ ID NO: 3) 10 μM 0.6 μL
Nuc gene amplification nucleic acid primer (SEQ ID NO: 4) 10 μM 0.6 μL
THUNDERBIRD (registered trademark) SYBR qPCR Mix (manufactured by Toyobo) 10 μL
Purified water 6.8 μL

Sample 2 μL

Real-time PCR conditions 95℃, 1 minute (1 cycle above)
95°C for 15 seconds 60°C for 30 seconds Fluorescence acquisition in steps of 60°C (40 cycles above)

Results The results of this example are shown in Table 1. Ct values were obtained for all sediment suspensions prepared from the blood culture solution, from those without dilution to those diluted 1,000,000 times, and it was confirmed that nucleic acid amplification occurred. The Ct value is a numerical value indicating the number of cycles when the amplification product reaches a certain amount, and generally, the Ct value tends to be lower in a reaction system having more target nucleic acids to be subjected to nucleic acid amplification. When a nucleic acid amplification inhibiting factor occurs, the Ct value becomes high.
When the Ct value in this example was confirmed, the Ct value was lower when the sediment suspension was diluted 10 to 1000 times than when it was not diluted. One possible cause of this phenomenon is considered to be the presence of substances that inhibit nucleic acid amplification, such as residues of blood components and activated carbon contained in blood culture bottles. The results of this example show that by appropriately diluting the sediment suspension, the above-mentioned inhibitory substances are also diluted, and as a result, the amplification efficiency can be further improved.
In addition, in this example, the step (II′) was inserted once to prepare a sample, and it was shown that the sample can be used as a target of nucleic acid analysis.

[Comparative Example 1: Direct nucleic acid analysis from blood culture solution]
(1) Preparation of sample The blood culture solution used in Example 1 was used as it was.
(2) Nucleic acid amplification The following reagents were added to the above-mentioned samples or purified water, respectively, and the methicillin resistance gene (mecA gene) was detected by the PCR method. Nucleic acid amplification was performed under the following conditions. GeneAmp PCR System 9700 (manufactured by Applied Biosystems) was used for nucleic acid amplification.

(3) Detection of Nucleic Acid Amplification Product The nucleic acid amplification product was confirmed by electrophoresis using 1×TAE agarose gel. The electrophoresis was performed at 100 V for 30 minutes, the agarose gel after the electrophoresis was stained with ethidium bromide, and the amplified product was visualized and confirmed by irradiation with ultraviolet rays.

Reagent sample or purified water Same as Example 1

Conditions for nucleic acid amplification Same as in Example 1

Results When the blood culture solution was directly used as the sample for nucleic acid analysis, the nucleic acid amplification product was not visualized.

[Example 3: Detection evaluation when liquid for suspending sediment is changed]
(1) Preparation of sample 200 μL of blood culture solution in which Staphylococcus aureus was cultured and 800 μL of purified water were mixed, and the mixture was centrifuged at 3000 g for 3 minutes to remove the supernatant and leave only the precipitate. After 900 μL of purified water was added to the precipitate to suspend it, the precipitate was centrifuged again, and the precipitate was suspended in 500 μL of each of the five kinds of liquids listed in (A) below. These suspensions were used as samples.
(A) Purified water Tris-HCl buffer (20 mM, pH 7.5)
Tris-HCl buffer (20 mM, pH 8.3)
Potassium hydroxide solution (20 mM)
Sodium hydroxide solution (20 mM)
(2) Real-time PCR method The following reagents were added to the above samples, respectively, and the methicillin resistance gene (mecA gene) was detected using the real-time PCR method. Detection was confirmed by calculation of Ct value. Real-time PCR was performed under the following conditions. Rotor-Gene Q (manufactured by Qiagen) was used for real-time PCR.

Reagents A 20 μL solution containing the following reagents was prepared.
Nucleic acid primer for amplifying mecA gene (SEQ ID NO: 1) 0.6 μL of 10 μM
Nucleic acid primer for amplification of mecA gene (SEQ ID NO: 2) 10 μM 0.6 μL
THUNDERBIRD (registered trademark) SYBR qPCR Mix (manufactured by Toyobo) 10 μL
Purified water 6.8 μL

Sample 2 μL

Real-time PCR conditions 95℃, 1 minute (1 cycle above)
Fluorescence acquisition in steps of 95°C for 15 seconds 60°C for 45 seconds 60°C (40 cycles above)

Results The results of this example are shown in Table 2. Ct values showing amplification of the mecA gene were obtained from the prepared sediment suspensions for all of the five kinds of liquids used this time. Therefore, it was shown that the prepared precipitate can be used as a sample for nucleic acid analysis regardless of which solution is used to prepare the precipitate.
Comparing the Ct values when individual sediment suspensions were used as samples, purified water showed the best results, and thereafter potassium hydroxide, Tris buffer (pH 8.3), Tris buffer (PH 7.5), followed by sodium hydroxide.

According to the present invention, microorganisms grown in blood culture can be detected more quickly and more accurately than in the prior art, which contributes to the fields of diagnosis and medical treatment.

Claims (5)

A sample preparation method for nucleic acid analysis comprising only the following steps (I) to (IV):
(I) A liquid containing no surfactant is added to and mixed with the blood culture liquid at a ratio of 1:3 to 1:6 of the blood culture liquid and the liquid containing no surfactant to prepare a suspension. Process,
(II) a step of centrifuging the suspension prepared in step (I) to remove the supernatant from the solution separated into a supernatant and a sediment,
(III) A step of adding a liquid containing no surfactant to the sediment remaining in the step (II) and mixing them to prepare a suspension,
(IV) A step of diluting the sediment suspension prepared in the step (III) 10 to 1000 times with a liquid containing no surfactant,
Here, the liquid containing no surfactant is any one or more selected from the group consisting of water, Tris buffer, Good buffer, potassium hydroxide solution, and sodium hydroxide solution, and a sample for nucleic acid analysis. Preparation method. The method according to claim 1, wherein after the step (III), a cycle of performing the following step (II′) and then performing the step (III) is inserted once or plural times.
(II') A step of centrifuging the suspension prepared in the step (III) to remove the supernatant from the solution separated into the supernatant and the sediment. The method according to claim 1 or 2, wherein in step (IV), the precipitate suspension prepared in step (III) is diluted 10 to 100 times with a liquid containing no surfactant. Free liquid surfactant is water, the method according to any one of claims 1-3. Analyzing the nucleic acid contained in a sample for nucleic acid analysis were prepared by the method of any of claims 1-4, the nucleic acid analysis method.