JP2017192350A - Method for lysis of mycoplasma pneumoniae and method for detection thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for lysing Mycoplasma pneumoniae from a biological sample to easily and in a short time release nucleic acids therefrom while preventing an inhibitor from eluting, even without the use of a special apparatus.SOLUTION: According to the present invention, a method comprises lysing Mycoplasma pneumoniae in sputum by coexisting Mycoplasma pneumoniae with an ionic surfactant (preferably sodium lauryl sulfate), purifying the RNA of Mycoplasma pneumoniae, and amplifying and detecting the purified RNA of Mycoplasma pneumoniae.SELECTED DRAWING: None

Description

  The present invention relates to a method for lysis of Mycoplasma pneumoniae and an inspection method using the same.

  Mycoplasma pneumoniae is a bacterial disease that is prevalent in the world, and not only its therapeutic method but also its diagnostic method is extremely important. Mycoplasma pneumoniae causes infections such as bronchitis and pneumonia (hereinafter called mycoplasma pneumonia) when it infects humans. Most of the variant pneumonia is mycoplasma pneumonia, and the frequency as community pneumonia is high. In Japan, the number of reports has increased from late autumn to early spring, and the affected age is mainly in early childhood, schoolchildhood, and adolescence.

  A more definitive diagnostic method for Mycoplasma pneumoniae is the culture method, but the growth rate of Mycoplasma pneumoniae is extremely slow, which can lead to delays in diagnosis, and hence the spread and development of secondary infections. Therefore, in parallel with the time-consuming culture method, a test method that can quickly obtain a result has been developed. Of particular interest are nucleic acid strand amplification and detection methods specific to Mycoplasma pneumoniae. This method is a method for determining the presence or absence of a bacterium by amplifying and detecting the nucleic acid chain of the bacterium using a primer specific for the nucleic acid (DNA, RNA, etc.) of the bacterium to be examined.

  In the diagnostic method to which the nucleic acid amplification method is applied, it is necessary to extract a nucleic acid chain by lysing Mycoplasma pneumoniae as a pretreatment. Examples of conventional lysis methods include chemical methods using organic solvents and surfactants (Patent Document 1), biological methods using enzymes, and ultrasonication or ultrasonic disruption in the presence of fine particles. There are physical methods such as repeated freezing and thawing. However, the conventional method takes time, and there is a problem that a biological sample containing Mycoplasma pneumoniae is excessively decomposed and a subsequent reaction is inhibited by a component derived from the biological sample.

JP 2014-97051 A

  The present invention provides a method for easily lysing mycoplasma pneumoniae while preventing elution of an inhibitor in a short time without using a special apparatus from a biological sample, and further minimizes damage to nucleic acid strands, particularly RNA. The object is to provide a method of release as a limit.

In order to achieve the above object, the present inventors have intensively studied to arrive at the present invention. That is, the present invention is as follows.
(1) A method for lysing Mycoplasma pneumoniae, wherein an ionic surfactant is allowed to coexist with Mycoplasma pneumoniae.
(2) The method according to (1), wherein the ionic surfactant is sodium lauryl sulfate.
(3) The method according to (1) or (2), which is a Mycoplasma pneumoniae contained in the soot.
(4) A method for purifying Mycoplasma pneumoniae RNA, which comprises lysing Mycoplasma pneumoniae by the method according to any one of (1) to (3) and then purifying the Mycoplasma pneumoniae RNA.
(5) A method for detecting Mycoplasma pneumoniae, comprising purifying Mycoplasma pneumoniae RNA by the method described in (4) and then amplifying and detecting the purified Mycoplasma pneumoniae RNA.

  The present invention is described in further detail below.

  In the present invention, an ionic surfactant is allowed to coexist in Mycoplasma pneumoniae and lysed. In the present invention, the presence form of Mycoplasma pneumoniae is not particularly limited, and examples thereof include those contained in a biological sample. Examples of the biological sample include sputum, pharyngeal wiping solution, nasopharyngeal wiping solution, and culture solution. As one aspect of the present invention, sputum is assumed as the biological sample. The method of the present invention does not solubilize inhibitory components in sputum so much, can lyse mycoplasma pneumoniae in sputum, and can prevent nucleic acid degradation.

  The ionic surfactant used in the present invention is not particularly limited as long as it can lyse Mycoplasma pneumoniae, and may be either a cationic surfactant or an anionic surfactant. For example, sodium lauryl sulfate, sodium cholate, hexadecyltrimethylammonium bromide, benzalkonium chloride, cetylpyridinium chloride, etc. can be used, preferably sodium lauryl sulfate, benzalkonium chloride, cetylpyridinium chloride, more preferably lauryl. Sodium sulfate. The concentration of the ionic surfactant is not particularly limited, but is preferably 1 to 20 w / v%, more preferably 2.5 to 10 w / v%.

  The present invention may further comprise an RNA purification step after lysis in this manner. Although it does not specifically limit as a purification method, Generally what can be applied to nucleic acid amplification is preferable, for example, AGPC method (Chomczynski, P., et al. (1987) Anal. Biochem., 162, 156-159.), Examples include the BOOM method (Japanese Patent No. 2680462). As a preferred embodiment of the BOOM method, a sample is added to a solution containing at least guanidine hydrochloride and 2-propanol, brought into contact with a solid phase containing silica, adsorbed to the solid phase containing silica, and then the nucleic acid is silica. And a method of eluting the nucleic acid in the absence of guanidine by washing under conditions where it is difficult to dissociate from the solid phase containing. The RNA of Mycoplasma pneumoniae to be purified is not particularly limited, and preferably ribosomal RNA (rRNA) can be mentioned.

  In the present invention, a person skilled in the art can appropriately select a nucleic acid amplification method for the RNA thus purified, and detect Mycoplasma pneumoniae. The nucleic acid amplification method is not particularly limited, and examples thereof include a PCR method, a NASBA method, and a TMA method, and preferably a TRC method that can amplify and measure RNA at a constant temperature, in one step, and in real time (for example, JP 2000-14400 A). No. 1, JP-A-2001-37500, JP-A-2001-353000, Ishiguro, T., et al. (2003) Anal. Biochem., 314, 1247-1252.).

  According to the present invention, an operator can lyse Mycoplasma pneumoniae in a short time, purify and extract nucleic acid containing RNA, and can be applied to various tests.

[Example 1] Preparation of oligonucleotide labeled with an intercalating fluorescent dye An oligonucleotide probe labeled with an intercalating fluorescent dye (hereinafter referred to as INAF probe) shown in the following (A) It was prepared based on the method disclosed in Japanese Patent No. 316587.
(A) via a linker between the 15th thymine and the 16th adenine from the 5 ′ end of the oligonucleotide consisting of SEQ ID NO: 1 (base sequence from GenBank No. NR — 070556.1 from the 2095th position to the 2112th position) Labeled with thiazole orange.

[Example 2] (Investigation of lysis efficiency and inhibition by ionic surfactant)
Mycoplasma pneumoniae lysis efficiency when using various ionic surfactants and reaction inhibition in the nucleic acid amplification process were examined. To 350 μL each of sodium lauryl sulfate with a final concentration of 1 w / v% and sodium cholate with a final concentration of 0.5 w / v%, 50 μL of sputum or 50 μL of water and 5 μL of Mycoplasma pneumoniae culture solution were added and vigorously stirred for 15 seconds. After stirring, centrifugation was performed at 10,000 g for 5 minutes, and 100 μL of the supernatant was added to a denaturing reagent consisting of 2.4 M guanidine thiocyanate, 15 mM sodium citrate, 60% 2-propanol. Thereafter, the denaturing reagent was brought into contact with the glass filter, the nucleic acid was captured by the glass filter, and the glass filter was washed with a washing solution 1 consisting of 200 mM potassium chloride and 40% 2-propanol. Next, the glass filter was washed with a washing solution 2 composed of 70% ethanol. Finally, after drying the glass filter, elution consisting of 10 mM 2-amino-2-hydroxymethyl-1,3-propanediol, 1 mM disodium ethylenediaminetetraacetate dihydrate, 0.01% sodium azide 50 μL of reagent was brought into contact with a glass filter to obtain 50 μL of eluate. The obtained eluate (hereinafter referred to as RNA sample) was measured by the following method.

(1) A reaction solution having the following composition was dispensed into an evaporation drying tube and evaporated to dryness.
Composition of reaction solution: Concentration is as follows. Final concentration of 60 mM Tris-HCl buffer (pH 8.35) after addition of RNA sample and starting solution (in 30 μL)
300 mM trehalose 0.48 mM each dATP, dCTP, dGTP, dTTP
Each 1.8 mM ATP, CTP, UTP
1.3 mM GTP
2.9 mM ITP
0.2 μM First primer: SEQ ID NO: 2 (T7 promoter added to the 5 ′ end of the complementary sequence of the 2171st to 2297th nucleotide sequences of GenBank No. NR — 07056.1)
0.2 μM Second primer: SEQ ID NO: 3 (base sequence from 1865th position to 1890th position of GenBank No. NR — 07056.1)
50 nM INAF probe (prepared in Example 1)
0.025 mg / mL bovine serum albumin 142U T7 RNA polymerase 6.4U AMV reverse transcriptase.

(2) After 15 μL of RNA sample was added after the above evaporation and drying, the mixture was kept at 46 ° C. for 5 minutes, and then 15 μL of an initial solution having the following composition was added and stirred.
Composition of starting solution: final concentration at the time of reaction (in 30 μL) 10.07% dimethyl sulfoxide 21 mM magnesium chloride 105 mM potassium chloride.

  (3) Using a fluorescence spectrophotometer with a temperature control function capable of directly measuring the evaporating and drying tube, the reaction solution was reacted at 46 ° C., and at the same time, the fluorescence intensity of the reaction solution was measured over time for 20 minutes. When the start solution is added and stirring is completed, the reaction time is 0 minutes, and the reaction mixture's fluorescence intensity ratio (the fluorescence intensity value of the predetermined time divided by the background fluorescence intensity ratio) exceeds 1.2. And the time at that time was defined as a positive time. The results are shown in Table 1. “ND” in Table 1 means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.2 or less (negative determination).

喀痰の代わりに水を加えた場合、ラウリル硫酸ナトリウム、コール酸ナトリウムのいずれも同程度の陽性時間で検出された。一方喀痰を加えた場合、コール酸ナトリウムではマイコプラズマニューモニエが検出されず、ラウリル硫酸ナトリウムのみで検出された。コール酸ナトリウムでは喀痰が過度に分解され、喀痰由来成分が核酸精製、核酸増幅を阻害したと考えられる。

When water was added instead of sputum, both sodium lauryl sulfate and sodium cholate were detected with similar positive times. On the other hand, when soot was added, Mycoplasma pneumoniae was not detected with sodium cholate, but only with sodium lauryl sulfate. It is considered that sodium cholate excessively decomposed the soot, and the soot-derived component inhibited nucleic acid purification and nucleic acid amplification.

[Example 3] (Examination of lysis efficiency by ionic surfactant)
Mycoplasma pneumoniae lysis efficiency when using various ionic surfactants and reaction inhibition in the nucleic acid amplification process were examined. To 400 μL each of sodium lauryl sulfate, benzalkonium chloride, and cetylpyridinium chloride having a final concentration of 5 w / v%, 100 μL and 10 μL of Mycoplasma pneumoniae culture solution were added and vigorously stirred for 15 seconds. After stirring, the mixture was centrifuged at 10,000 g for 5 minutes, and 100 μL of the supernatant was measured in the same manner as in Example 2.

ラウリル硫酸ナトリウムによる溶菌の効率が最も良好であることがわかる。

It can be seen that the efficiency of lysis with sodium lauryl sulfate is the best.

[Example 4] (Examination of sodium lauryl sulfate concentration)
100 μL of ナ ト リ ウ ム and 10 μL of Mycoplasma pneumoniae culture solution were added to 400 μL of sodium lauryl sulfate having a final concentration of 2.5 w / v%, 5 w / v%, and 10 w / v%, and vigorously stirred for 15 seconds. After stirring, the mixture was centrifuged at 10000 g for 5 minutes, and 150 μL of the supernatant was measured in the same manner as in Example 2.

どの濃度でもマイコプラズマニューモニエを検出出来ていることがわかる。

It can be seen that Mycoplasma pneumoniae can be detected at any concentration.

Claims (5)

  A method for lysing Mycoplasma pneumoniae, wherein an ionic surfactant is allowed to coexist with Mycoplasma pneumoniae.   The method of claim 1, wherein the ionic surfactant is sodium lauryl sulfate.   The method according to claim 1 or 2, which is a Mycoplasma pneumoniae contained in the cocoon.   A method for purifying Mycoplasma pneumoniae RNA, which comprises lysing Mycoplasma pneumoniae by the method according to any one of claims 1 to 3, and then purifying the Mycoplasma pneumoniae RNA.   A method for detecting Mycoplasma pneumoniae, comprising purifying Mycoplasma pneumoniae RNA by the method according to claim 4 and then amplifying and detecting the purified Mycoplasma pneumoniae RNA.