SAMPLING KIT USED IN DETERMINATION OF RESPIRATORY INFECTION

Abstract

Disclosed is a sampling kit for determining respiratory infection, the kit including: a first vessel containing a washing-out solution; and a second vessel containing a transport medium containing a deactivating agent, so that sampling corresponding to a pre-analytical stage in a protocol for determination of the presence or absence of respiratory infection pathogens can be attained easily, safely, and stably, and self-sampling of a respiratory infection pathogen can be attained.

Claims

1. A sampling kit for determining respiratory infection, the kit comprising: (a) a first vessel containing a washing-out solution for washing out a respiratory infection pathogen from the oral cavity and/or throat, the washing-out solution containing an aqueous solution; and (b) a second vessel containing a transport medium, the transport medium containing a deactivating agent for deactivating the respiratory infection pathogen.

2. The kit of claim 1, wherein the aqueous solution of the washing-out solution is a water- or saline-based solution.

3. The kit of claim 2, wherein the saline is phosphate buffered saline (PBS) or normal saline.

4. The kit of claim 3, wherein the saline is PBS.

5. The kit of claim 1, wherein the washing-out solution further contains at least one component selected from the group consisting of: (a) an alcohol selected from the group consisting of ethanol and isopropanol; and (b) a detergent.

6. The kit of claim 1, wherein the transport medium containing the deactivating agent comprises (i) a chaotropic agent.

7. The kit of claim 6, wherein the transport medium further contains at least one component selected from the group consisting of: (ii) a detergent; (iii) a reductant; and (iv) a chelator.

8. The kit of claim 6, wherein the transport medium serves as a deactivating function by lysis of the respiratory infection pathogen and a stabilizing function of a nucleic acid material released from the lysed pathogen.

9. The kit of claim 6, wherein the chaotropic agent is guanidine thiocyanate, guanidine isocyanate, or guanidine hydrochloride.

10. The kit of claim 7, wherein the detergent is sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, or N-lauroyl sarcosine.

11. The kit of claim 7, wherein the reductant is 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, dithiothreitol, or dimethylsulfoxide.

12. The kit of claim 7, wherein the chelator is ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, or lithium citrate.

13. The kit of claim 1, wherein sampling is attained by applying the washing-out solution of the first vessel to the human oral cavity and/or throat to perform gargling and placing a resultant liquid of gargling into the transport medium of the second vessel.

14. The kit of claim 13, wherein the kit is configured to sample a mouthwash sample.

15. The kit of claim 1, wherein sampling is attained by applying the washing-out solution of the first vessel to the human oral cavity and/or throat to perform (i) brushing or (ii) gargling and brushing, and placing a resultant liquid of brushing, a resultant liquid of gargling and a resultant liquid of brushing, or a brush into the transport medium of the second vessel.

16. The kit of claim 15, wherein the kit further comprises an oral brush.

17. The kit of claim 1, wherein the kit is a kit for self-sampling.

18. The kit of claim 1, wherein the kit is for determining infection with a respiratory virus and/or a respiratory bacterium.

19. The kit of claim 18, wherein the respiratory virus is an influenza virus, a respiratory syncytial virus (RSV), an adenovirus, an enterovirus, a parainfluenza virus (PIV), a metapneumovirus (MPV), a bocavirus, a rhinovirus, and/or a coronavirus.

20. The kit of claim 19, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

21. The kit of claim 1, wherein the product sampled by the kit is directly used in the separation of a nucleic acid without additional cell lysis.

22. The kit of claim 21, wherein the nucleic acid is separated using magnetic particles and the sampled product is applied to a binding buffer containing magnetic particles.

23. The kit of claim 1, wherein the kit is applied to non-aerosol generating sampling.

24. The kit of claim 1, wherein the kit is for obtaining a sample used for real-time nucleic acid amplification involving nucleic acid amplification.

25. A sampling method for determining respiratory infection, the method comprising: (a) placing a resultant liquid of gargling, which is obtained by applying a washing-out solution containing an aqueous solution to the oral cavity and/or throat and performing gargling, into a transport medium containing a deactivating agent for deactivating a respiratory infection pathogen.

26. A nucleic acid molecule extraction method for determining respiratory infection, the method comprising: (a) applying a resultant product of the method of claim 25 to a binding buffer containing magnetic particles and binding nucleic acid molecules in the resultant product to the magnetic particles; and (b) subjecting the nucleic acid molecules bound to the magnetic particles to elution, thereby extracting the nucleic acid molecules for respiratory infection determination.

27. A method for determining respiratory infection, the method comprising: (a) applying nucleic acid molecules, extracted by the method of claim 26, to a real-time amplification solution; and (b) detecting a signal, generated by performing real-time amplification using the real-time amplification solution, to determine the presence or absence of respiratory infection.

Description

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

[0077] Hereinafter, the present disclosure will be described in detail through examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

Example 1

Respiratory Infection Pathogen Detection 1 Using Washing-Out Sample Collection Manner

[0078] The present inventors collected samples from the respiratory organ in a washing-out manner (i.e., gargling manner) but not a swab manner, and investigated whether the presence or absence of a respiratory infection pathogen was detectable. [0079] (1) Collection of Samples

[0080] Samples were collected in a washing-out manner from ten patients infected with influenza A virus. Each patient gargled with 4 mL of saline so as to collect samples in the oral cavity and throat. As for a control, samples were collected from ten non-infected subjects in the same manner above. [0081] (2) Deactivation of Gargle Samples

[0082] Each gargle sample was spat into an empty vessel, and the gargle sample was mixed with 2 mL of a transport medium solution containing a lysis component (3 M guanidine thiocyanate, 0.5 wt % sodium dodecyl sulfate, and 120 mM Tris buffer, pH 7.2). [0083] (3) Extraction of Nucleic Acid

[0084] The extraction of nucleic acid was carried out using the STARMag 96X4 Universal Cartridge Kit (Cat. No. 744300.4.UC384, Seegene Inc.) and the automatic nucleic acid extraction system Microlab NIMBUS (Cat. No. 65415-02, Hamilton). The extraction was carried out according to the manufacturer of an extraction reagent and the operation manual of the system.

[0085] 300 μL of the deactivated sample contained in the transport medium was placed into the automatic nucleic acid extraction system.

[0086] The extraction reagent contains a lysis buffer, proteinase K, a binding buffer, washing buffer 1, washing buffer 2, washing buffer 3, a elution solution, and magnetic beads. [0087] (4) Detection of Nucleic Acid

[0088] The presence or absence of the pathogen was tested using the resultant product of nucleic acid extraction.

[0089] A reaction mixture for detection was prepared using Allplex™ respiratory panel 1 Assay reagent (Cat. No. RP9801X, Seegene, Inc.) and the automatic nucleic acid extraction system Microlab NIMBUS (Cat. No. 65415-02, Hamilton). The extraction was carried out according to the manufacturer of an extraction reagent and the operation manual of the system. An internal control (IC) provided from the reagent manufacturer was used according to a protocol.

[0090] The reaction mixture was prepared by mixing 5 μL of 5×RP1 MOM, 5 μL of RNase-free Water, 5 μL of 5×Real-time One-Step Buffer, 2 μL of Real-time One-Step Enzyme, and 8 μL of a nucleic acid extract.

[0091] PCR plates containing the reaction mixture were subjected to real-time PCR using a real-time PCR machine (CFX96 Real-time cycler, Bio-Rad, US).

[0092] The amplification was conducted by reaction at 50° C. for 20 min, reaction 95° C. for 15 min, and 45 cycles of reaction at 95° C. for 10 sec, at 60° C. for 60 sec, and at 72° C. for 10 sec. The fluorescence was measured at 60° C. and 72° C. every cycle.

[0093] Data were analyzed by Seegene Viewer S/W (Seegene, Korea).

[0094] As a result of analysis, nine samples out of ten patient samples showed amplification results exceeding the threshold at a Ct value between 25 and 32, each indicating a positive result on the respiratory pathogen. Ten out of ten non-infected subjects showed negative results. That is, the washing-out sampling manner can exhibit a clinical sensitivity of 90% and a clinical specificity of 100%, and is very effective in the detection of respiratory pathogens.

Example 2

Respiratory Infection Pathogen Detection 2 Using Washing-Out Sample Collection Manner

[0095] The present inventors investigated whether the presence or absence of a respiratory infection pathogen was detectable by using an extraction manner without separate lysis in the nucleic acid extraction procedure while a pathogen was deactivated using a transport medium solution containing a lysis component.

[0096] The same samples, reagents, and systems as in Example 1 were used except for the following changes in the extraction procedure. That is, the extraction procedure of the extraction system in Example 1 was changed such that, instead of mixing with the lysis buffer, a binding buffer having the same volume as a lysis buffer to be used was additionally mixed.

[0097] The preparation and detection of the reaction mixture for detection were also carried out in the same manner as in Example 1.

[0098] As a result of testing, nine samples out of ten patient samples showed amplification results exceeding the threshold at a Ct value between 30 and 38, each indicating a positive result on the respiratory pathogen. Ten samples out of ten non-infected subjects showed negative results. That is, when samples were collected in the washing-out sampling manner of the present disclosure and the deactivating transport solution contains a lysis component, such a method exhibited a clinical sensitivity of 90% and a clinical specificity of 100%, even without additional lysis in the extraction procedure, and is very effective in the detection of respiratory pathogens.

Comparative Example 1

Respiratory Infection Pathogen Detection Using Swab Sample Collection Manner

[0099] As for a control, samples were collected from respiratory organs in a swab manner. [0100] (1) Collection of Samples

[0101] Samples were collected by inserting a swap into the nose of the same patient as in Example 1 above. [0102] (2) Preservation of Swab Samples

[0103] Each swab was stored in 3 mL of PBS solution. [0104] (3) Extraction and Detection of Nucleic Acid

[0105] Extraction and detection were conducted using the same reagents and systems as in the method described in Example 1 above. 300 μL of the samples contained in the transport medium were placed into the automatic nucleic acid extraction system.

[0106] As a result of analysis, ten out of ten patient samples showed amplification results exceeding the threshold at a Ct value between 22 and 30, each indicating a positive result on respiratory pathogens. Ten samples out of ten non-infected subjects showed negative results. That is, the conventional swab sampling manner exhibited a clinical sensitivity of 100% and a clinical specificity of 100%.