KR20230118173A - Sampling Swab Tool and Sampling Method for Detecting Respiratory Pathogens - Google Patents

Abstract

The present invention relates to a sample collection swab tool and a method for detecting respiratory pathogens. In the present invention, the overall length of the sample collection swap tool is reduced to a specific length, or the ratio of the sum of the lengths of the head portion and the support portion to the length of the gripping portion constituting the sample collection swap tool is adopted as a specific value, It is possible to accurately and easily collect a nasal swab sample and / or an oral swab sample from a sample collection site (specifically, the anterior nasal cavity and / or oral cavity), collect a sufficient amount of sample to detect a respiratory pathogen, and It can be used for self-sampling by non-specialists as well as sampling by non-specialists, and respiratory pathogens can be detected from collected nasal swab samples and oral swab samples.

Description

Sampling Swabs and Sampling Methods for Detecting Respiratory Pathogens

Cross reference to related applications

This patent application claims priority to Korean Patent Application No. 2021-0019204 filed with the Korean Intellectual Property Office on February 10, 2021, and the disclosures of the above patent applications are incorporated herein by reference.

technology field

The present invention relates to a sample collection swab tool and a method for detecting respiratory pathogens.

Respiratory diseases cause significant mortality, especially in children, the elderly and immunocompromised people. It is very important to identify the causative pathogen for infection control and appropriate patient management. Respiratory pathogen testing has developed rapidly with the development of molecular diagnostic technology, especially real-time PCR. Real-time PCR is more sensitive than traditional methods, and can detect a variety of pathogens simultaneously.

Various upper respiratory tract (URT) samples and lower respiratory tract samples are used for respiratory pathogen testing by molecular diagnosis.

In general, as upper respiratory tract samples, nasopharyngeal swab specimens and oropharyngeal swab specimens are widely used. Meanwhile, sputum and bronchoalveolar lavage (BAL) samples are used as lower respiratory tract samples (Falsey et al., 2012; Branche et al., 2014; Jeong et al., 2014).

Recently, as SARS-CoV-2 is prevalent worldwide, real-time PCR diagnostic kits are in the spotlight. For the diagnosis of coronavirus, nasopharyngeal swab samples and oropharyngeal swab samples are used as upper respiratory tract samples, and sputum and bronchial lavage fluid are used as lower respiratory tract samples. The nasopharyngeal swab sample and oropharyngeal swab sample collect secretions from the nasopharynx and oropharynx, respectively, using a cotton swab and store them in a transport medium. and keep

However, lower respiratory tract samples are difficult to collect and may require pre-treatment prior to nucleic acid extraction, and in particular, sputum samples are not applicable in the early stages of infection when the patient exhibits practically no cough symptoms or a dry cough. Sampling by nasopharyngeal swab and oropharyngeal swab has limitations that must be performed by experts and is risky as it is an aerosol-generating sampling method.

In the sampling method by nasopharyngeal swab and oropharyngeal swab performed by experts, as shown in FIG. However, this method was not suitable as a self-sampling method in which non-specialists collect samples by themselves and transport the samples to inspection institutions.

On the other hand, not only experts but also non-experts were using nasopharyngeal swabs and oropharyngeal swabs as shown in A in FIG. 1 even when collecting nasal swab samples and oral swab samples. However, since force is not properly transmitted to the swab, a sufficient amount of sample required to detect respiratory pathogens cannot be collected, or the total length of the swab is long even though a total nasal swab sample and an oral swab sample must be collected, so that the nasopharynx There was a problem with insertion to the posterior wall and the posterior wall of the oropharynx.

Therefore, the inventors of the present invention are working on the development of a technology that can easily collect a respiratory sample and collect an amount of sample necessary to detect respiratory pathogens not only in the case of sampling by experts but also in the case of self-sampling by non-experts. recognized the need.

Throughout this specification, numerous citations and patent documents are referenced and the citations are indicated. The disclosure contents of the cited documents and patents are hereby incorporated by reference in their entirety to more clearly describe the content of the present invention and the level of the technical field to which the present invention belongs.

The present inventors have tried to overcome the above-mentioned problems of the conventional sample collection swap tool and develop a sample collection swap tool that can effectively collect an anterior nasal swab sample and/or an oral swab sample. As a result, the inventors of the present invention have prepared a sample collection swap tool by reducing the overall length of a sample collection swap tool used for collecting a conventional nasal cavity and/or oral swab sample to a specific length, or a part constituting the sample collection swap tool. As a result of developing a sample collection swap tool that adopts the ratio of the sum of the lengths of the head part and the support part to the length of the branch as a specific value, the sample collection site (specifically, the anterior nasal cavity and / or oral cavity) can be accurately and easily A total nasal swab sample and/or an oral swab sample can be taken, a sample amount sufficient to detect respiratory pathogens can be collected, and it can be used for self sampling by non-experts as well as sampling by experts. The present invention was completed by confirming that it can be used and that respiratory pathogens can be detected from the collected samples.

Accordingly, an object of the present invention is to provide a sample collection swab tool.

Another object of the present invention is to provide a sampling kit for detecting respiratory pathogens.

Another object of the present invention is to provide a sampling method for detecting respiratory pathogens.

Other objects and advantages of the present invention will become more apparent with the following embodiments, claims and drawings.

According to one aspect (first aspect) of the present invention, there is provided a sample collection swap tool comprising:

(a) a supporting part having a cut groove formed on an outer circumferential surface of one end thereof;

(b) a head part having a fiber layer formed at the other end of the support part to collect samples; and

(c) a gripping part extending from the cut groove of the upper limb support; And, the total length of the swap tool is 90-120 mm.

According to another aspect (second aspect) of the present invention, there is provided a sampling swab tool comprising:

(a) a supporting part having a cut groove formed on an outer circumferential surface of one end thereof;

(b) a head part having a fiber layer formed at the other end of the support part to collect samples; and

(c) a gripping part extending from the cut groove of the upper limb support; And, the sum of the lengths of the head part and the support part and the length of the gripping part are 1.4-3.0:1.

The present inventors have tried to overcome the above-mentioned problems of the conventional sample collection swap tool and develop a sample collection swap tool that can effectively collect an anterior nasal swab sample and/or an oral swab sample. As a result, the inventors of the present invention have prepared a sample collection swap tool by reducing the overall length of a sample collection swap tool used for collecting a conventional nasal cavity and/or oral swab sample to a specific length, or a part constituting the sample collection swap tool. As a result of developing a sample collection swap tool that adopts the ratio of the sum of the lengths of the head part and the support part to the length of the branch as a specific value, the sample collection site (specifically, the anterior nasal cavity and / or oral cavity) can be accurately and easily A total nasal swab sample and/or an oral swab sample can be collected, a sample amount sufficient to detect respiratory pathogens can be collected, and it can be used for self sampling by non-experts as well as sampling by experts. It was confirmed that it can be used and that respiratory pathogens can be detected from the collected samples.

Specimens collected using the swab tool of the present invention are respiratory specimens, and specifically, the respiratory specimens are nasal specimens and/or oral cavity specimens.

The present invention is characterized in that it enables self-sampling by non-experts as well as sampling by experts.

According to one embodiment of the present invention, the sampling swap tool is a self sampling tool.

Hereinafter, the present invention will be described in detail through exemplary drawings. In adding reference numerals to components of each drawing, it should be noted that the same components have the same numerals as much as possible even if they are displayed on different drawings. In addition, in describing the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description will be omitted.

1B to D show a sampling swab tool used to collect an anterior nasal swab specimen and/or an oral swab specimen according to one embodiment of the present invention.

Referring to B to D of FIG. 1 , the sample collection swap tool 100 of the present invention includes (a) a supporting part 10 and (b) a head part 11 ) and (c) a gripping part (13).

The supporting part 10 is a part that supports the sample collection swab tool as a whole, and has a cutting groove 12 formed at one end thereof.

The head part 11 is a part that directly contacts the sample collection site, and a fiber layer is formed at the other end of the support section to collect samples.

The gripping part 13 is a part that the sample collector holds by hand, and allows the sample collector's force to be transmitted to the head through the support to collect the sample through the head.

1A shows an example of applying a swab tool used as a conventional nasopharyngeal swab tool to a nasal swab tool and an oral swab tool.

Referring to A of FIG. 1, the swab tool 1 of A of FIG. 1 has a total length of 157 mm, and when a sample is collected from the anterior nasal cavity and/or oral cavity (see FIGS. 3A and 3B) using this swab tool 1. It is difficult to accurately sample in the anterior nasal cavity and oral cavity, and even inserted into the posterior wall of the nasopharynx and oropharynx, and the force control for sampling is not smooth. There is a difficult problem.

Accordingly, the inventors of the present invention intend to solve the above-described problems of the conventional nasal swab tool and oral swab tool by reducing the overall length compared to the conventional nasopharyngeal swab tool.

For this purpose, the overall length of the sampling swab tool 100 of the present invention was adopted as 90-120 mm, which is one of the characteristics of the present invention.

When the total length exceeds 120 mm, the force control for the swab tool is not smooth, making it difficult to perform accurate sampling in the anterior nasal cavity and oral cavity, which are sampling sites, and there is a disadvantage in that the amount of sample required for the examination cannot be collected. If the total length is less than 90 mm, there is a problem in that the wall of the nasal cavity or the oral cavity may be damaged due to the transfer of excessive force by the sample collector during sampling.

Specifically, the total length of the sample collection swap tool 100 is 90-119 mm, 90-118 mm, 90-116 mm, 90-114 mm, 90-112 mm, 90-110 mm, 90-108 mm, 90-106 mm, 90-104 mm, 90-102 mm, 90-100 mm, 90-98 mm, 90-96 mm, 90-95 mm, 92-119 mm, 92-118 mm, 92-116 mm, 92-114 mm, 92-112 mm, 92-110 mm, 92-108 mm, 92-106 mm, 92-104 mm, 92-102 mm, 92-100 mm, 92-98 mm, 92-96 mm, 92-95 mm, 93-119 mm, 93-118 mm, 93-116 mm, 93-114 mm, 93-112 mm, 93-110 mm, 93-108 mm, 93-106 mm, 93-104 mm, 93-102 mm, 93-100 mm, 93-98 mm, 93-96 mm, 93-95 mm, 94-119 mm, 94-118 mm, 94-116 mm, 94-114 mm, 94-112 mm, 94-110 mm, 94-108 mm, 94-106 mm, 94-104 mm, 94-102 mm, 94-100 mm, 94-98 mm, 94-96 mm, 110-119 mm, 110-118 mm, 110-116 mm, 110-115 mm, 112-119 mm, 112-118 mm, 112-116 mm, 112-115 mm, 113-119 mm, 113-118 mm, 113-116 mm, 113-115 mm, 114-119 mm, 114-118 mm, 114-116 mm, 114-115 mm, 95 mm or 114 mm.

Specifically, the length of the support 10 is 60-68 mm, 60-67 mm, 60-66 mm, 60-64 mm, 60-63 mm, 60-62 mm, 61-68 mm, 61-67 mm, 61-66 mm, 61-64 mm, 61-63 mm, 61-62 mm, 62-68 mm, 62-67 mm, 62-66 mm, 62-64 mm, 62-63 mm, 64-68 mm, 64-67 mm, 64-66 mm, 65-68 mm, 65-67 mm, 65-66 mm, 66-68 mm, 66-67 mm, 47 mm or 66 mm.

When the support portion has the length, the sample collection swab tool may be supported as a whole.

Specifically, the length of the head portion 11 is 13-17 mm, 13-16 mm, 13-15 mm, 13-14 mm, 14-17 mm, 14-16 mm, 14-15 mm, 15-17 mm , 15-16 mm, 16-17 mm, or 15 mm.

When the head portion has the above length, the sample can be collected by directly contacting the sample collection site.

Specifically, the length of the gripping portion 13 is 28-38 mm, 28-36 mm, 28-34 mm, 30-38 mm, 30-36 mm, 30-34 mm, 32-38 mm, 32-36 mm , 32-34 mm, 34-38 mm, 34-36 mm, or 33 mm.

When the gripping part has the above length, the specimen collector's force is transmitted to the head part through the support part, allowing the specimen to be collected through the head part.

In addition, referring to A of FIG. 1, the swap tool 1 of A of FIG. 1 has a total length of the head part and the support part of 81 mm and a length of the holding part of 76 mm, and the length of the head part and the support part is 81 mm. The ratio of the sum of and the length of the gripping part is 1.1:1. In this case, since the length of the gripping part does not differ from the sum of the lengths of the head part and the support part, when a sample is collected from the anterior nasal cavity and/or oral cavity (see FIGS. 3a and 3b) using such a swap tool, the anterior nasal cavity and It is difficult to accurately sample in the oral cavity, and it is even inserted into the posterior wall of the nasopharynx and oropharynx, and the force control for sampling is not smooth, making it difficult to collect the required amount of sample for molecular diagnosis (specifically, molecular diagnosis through PCR). .

Accordingly, the inventors of the present invention reduce the ratio of the length of the gripping part to the sum of the lengths of the head part and the support part more than the conventional nasopharyngeal swab tool, to solve the above-mentioned problems of the conventional nasal swab tool and oral swab tool. do.

For this purpose, the sum of the lengths of the head part 11 and the support part 10 and the length of the grip part 13 included in the sample collection swab tool 100 of the present invention were adopted at a ratio of 1.4-3.0: 1 , which is another one of the features of the present invention.

When the length of the gripping part is made shorter than the sum of the lengths of the head part and the support part, that is, when the sum of the lengths of the head part and the support part and the length of the gripping part are set to 1.4-3.0 : 1, the force adjustment for the swap tool is It is smooth, so it is possible to accurately sample from the anterior nasal cavity and oral cavity, which are sampling sites, and to collect the amount of sample required for examination.

Specifically, the sum of the lengths of the head part 11 and the support part 10 and the length of the gripping part 13 are 1.4-2.9 : 1, 1.4-2.8 : 1, 1.4-2.6 : 1, 1.4-2.5 : 1 , 1.4-2.4:1, 1.4-2.2:1, 1.4-2.0:1, 1.4-1.9:1, 1.6-2.9:1, 1.6-2.8:1, 1.6-2.6:1, 1.6-2.5:1, 1.6 -2.4:1, 1.6-2.2:1, 1.6-2.0:1, 1.6-1.9:1, 1.8-2.9:1, 1.8-2.8:1, 1.8-2.6:1, 1.8-2.5:1, 1.8-2.4 :1, 1.8-2.2:1, 1.8-2.0:1, 1.8-1.9:1, 1.9-2.9:1, 1.9-2.8:1, 1.9-2.6:1, 1.9-2.5:1, 1.9-2.4:1 , 1.9-2.2:1, 1.9-2.0:1, 2.0-2.9:1, 2.0-2.8:1, 2.0-2.6:1, 2.0-2.5:1, 2.0-2.4:1, 2.0-2.2:1, 2.2 -2.9:1, 2.2-2.8:1, 2.2-2.6:1, 2.2-2.5:1, 2.2-2.4:1, 2.4-2.9:1, 2.4-2.8:1, 2.4-2.6:1, 2.4-2.5 : 1, 2.6-2.9: 1, or 2.6-2.8: 1.

More specifically, the lengths of the support part 10, the head part 11 and the gripping part 13 are 3-5: 1: 2-2.8. According to this embodiment, by configuring the length of the gripping part to be shorter than the length of the supporting part, it is possible to easily collect the whole nasal cavity and/or oral swab sample. Specifically, the lengths of the support part, the head part, and the gripping part are 3-5: 1: 2.0-2.6, 3-5: 1: 2.0-2.4, 3-5: 1: 2.0-2.3, 3-5: 1: 2.1-2.8, 3-5: 1 : 2.1-2.6, 3-5: 1 : 2.1-2.4, 3-5: 1 : 2.1-2.3, 3-5: 1 : 2.2-2.8, 3-5: 1 : 2.2-2.6, 3-5: 1 : 2.2-2.4, 3-5: 1 : 2.2-2.3, 3-5: 1 : 2.3-2.8, 3-5: 1 : 2.3-2.6, 3-5: 1 : 2.3-2.4, 3-5: 1 : 2.4-2.8, 3-5: 1 : 2.4-2.6, 3.1-5: 1 : 2.0-2.8, 3.1-4.7: 1 : 2.0-2.8, 3.1-4.5: 1 : 2.0-2.8, 3.1-4.2: 1 : 2.0-2.8, 3.1-3.9: 1 : 2.0-2.8, 3.1-3.6: 1 : 2.0-2.8, 3.1-3.3: 1 : 2.0-2.8, 3.2-5: 1 : 2.0-2.8, 3.2-4.7: 1 : 2.0-2.8, 3.2-4.5: 1 : 2.0-2.8, 3.2-4.2: 1 : 2.0-2.8, 3.2-3.9: 1 : 2.0-2.8, 3.2-3.6: 1 : 2.0-2.8, 3.2-3.3: 1 : 2.0-2.8, 3.3-5: 1 : 2.0-2.8, 3.3-4.7: 1 : 2.0-2.8, 3.3-4.5: 1 : 2.0-2.8, 3.3-4.2: 1 : 2.0-2.8, 3.3-3.9: 1 : 2.0-2.8, 3.3-3.6: 1 : 2.0-2.8, 4.0-5: 1 : 2.0-2.8, 4.0-4.8: 1 : 2.0-2.8, 4.0-4.6: 1 : 2.0-2.8, 4.0-4.5: 1 : 2.0-2.8, 4.2-5: 1 : 2.0-2.8, 4.2-4.8: 1 : 2.0-2.8, 4.2-4.6: 1 : 2.0-2.8, 4.2-4.5: 1 : 2.0-2.8, 4.4-5: 1 : 2.0-2.8, 4.4-4.8: 1 : 2.0-2.8, 4.4-4.6: 1 : 2.0-2.8.

According to one embodiment of the present invention, in the sample collection swap tool 100 of the present invention, the total length of the sample collection swap tool is 90-120 mm, and the sum of the lengths of the head part 11 and the support part 10 and the length of the gripping part 13 is 1.4-3.0:1.

According to another embodiment of the present invention, the support portion 10 includes a display portion 14 displaying the limiting length of nasal insertion at the distal end of the support portion 10 adjacent to the head portion 11 .

If a sample is to be collected from the nasopharynx and oropharynx using the swab tool, the swab tool is used to collect the sample from the nasopharynx and the posterior wall of the oropharynx, so there is no need to consider the length of insertion of the swab tool. However, when it is desired to collect nasal and/or oral swab specimens (particularly, nasal swab specimens), precise positioning of the head of the swab tool at the sampling site is required for self-sampling by non-experts as well as sampling by experts. This is important. For this reason, the sample collection swab tool 100 of the present invention includes a display unit 14 displaying the limiting length of nasal insertion at the end of the support unit 10 adjacent to the head unit 11 . Through this display unit 14, the specimen collector can properly induce the insertion of the specimen collection swab tool, thereby effectively collecting the nasal cavity swab specimen.

More specifically, the display unit 14 includes grooves, protrusions, color bands, or a combination thereof formed at predetermined intervals on the outer circumferential surface of the support unit. More specifically, the display unit includes grooves formed at predetermined intervals, color bands, or a combination thereof, and most specifically includes color bands formed at predetermined intervals.

More specifically, the color band includes a color line.

The predetermined interval may be determined in consideration of a guideline related to nasal swab sample collection, or may be determined in consideration of the size or length of the anterior nasal cavity. The predetermined interval may be determined in consideration of various factors such as age, height, face size or nose size. For example, according to guidelines for age-specific nasal insertion lengths, the length from the distal end of the head is 15 mm for ages 2 to 6 years, 20 mm for ages 6 to 12 years, and for those over 12 years of age. In some cases, it is set to 30 mm.

The predetermined interval may be selected from 1 to 40 mm from an end of the head portion that is not adjacent to the support portion or from an end of the head portion adjacent to the support portion, and specifically may be selected from 1 to 40 mm from an end of the head portion adjacent to the support portion. And, for example, it may be 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm or 15 mm. Alternatively, the predetermined spacing may be the same or different. For example, the first interval from the end of the head portion adjacent to the support portion may be set to 5 mm, the second interval to 5 mm from the first interval, and the third interval from the second interval to 5 mm, or A first distance from the end of the head portion adjacent to 5 mm, a second distance of 3 mm from the first distance, and a third distance from the second distance may be set to 6 mm.

A plurality of grooves, protrusions, color bands, or a combination thereof included in the display unit 14 may be included considering the predetermined interval. For example, the number of grooves, protrusions, color bands, or combinations thereof may be 1 to 5, 2 to 5, 2 to 4, or 3 to 4.

Referring to B of FIG. 1 , the display unit 14 includes three color bands. The first color band extends 15 mm from the end of the head not adjacent to the support, the second color band extends 5 mm from the first color band, and the third color band extends 10 mm from the second color band. formed far away.

The three color bands may be distinguished by displaying all different colors or alternately displaying different colors so as to indicate the limiting length of nasal insertion. For example, you might want the first color band to be red, the second color band green, and the third color band blue, or the first and third color bands red, and the second color band blue. can

The colors shown in the color bands include a variety of colors known in the art.

More specifically, the display unit 14 displays the limit length of nasal insertion for each age group.

Referring to B of FIG. 1, for example, according to the guideline for limiting the length of nasal insertion by age, the limiting length from the end of the head is 15 mm for 2 to 6 years old and 20 mm for 6 to 12 years old. mm, and if it is 30 mm in the case of over 12 years old, a red color band from the end of the head to 15 mm, a blue color band from the red color band to 5 mm, and a blue color band from the blue color band to 10 mm By forming a green color band, it is possible to display the limited length of nasal insertion for each age group on the display unit.

2 shows the head portion 11 of the sample collection swab tool 100 according to one embodiment of the present invention.

The fiber layer formed to collect samples in the head part can be formed by various methods known in the art, for example, it can be formed in the head part by a flocking method. According to the flocking method, adhesive may be applied to the surface of the head portion and small fiber yarns may be vertically deposited in an electrostatic field.

According to another embodiment of the present invention, the thickness of the fiber layer formed on the head part is 0.2-3 mm. Specifically, the thickness of the fiber layer is 0.2-2.8 mm, 0.2-2.5 mm, 0.2-2.2 mm, 0.2-1.8 mm, 0.2-1.5 mm, 0.2-1.2 mm, 0.2-0.8 mm, 0.4-2.8 mm, 0.4-2.5 mm, 0.4-2.2 mm, 0.4-1.8 mm, 0.4-1.5 mm, 0.4-1.2 mm, 0.4-0.8 mm, 0.6-2.8 mm, 0.6-2.5 mm, 0.6-2.2 mm, 0.6-1.8 mm, 0.6-1.5 mm, 0.6-1.2 mm, 0.6-0.8 mm, 1.0-2.8 mm, 1.0-2.5 mm, 1.0-2.2 mm, 1.0-1.8 mm, 1.0-1.5 mm, 1.0-1.2 mm, 1.2-2.8 mm, 1.2-2.5 mm, 1.2-2.2 mm, 1.2-1.8 mm, 1.2-1.5 mm, 1.5-2.8 mm, 1.5-2.5 mm, 1.5-2.2 mm, 1.5-1.8 mm, 2.2-2.8 mm, 2.2-2.5 mm, 2.2-2.4 mm, 2.4-2.8 mm, or 2.4-2.5 mm.

The fibers of the fibrous layer may use fibers of various lengths. For example, when forming a fiber layer by a flocking method, fibers of the same length are used to form a uniform thickness. For example, the fiber layer may be formed using fibers having a length of 0.6 to 3 mm. When the fiber layer is formed using fibers having such a length, the thickness of the fiber layer formed on the head part is 0.6-3 mm.

More specifically, the head portion 11 has an elliptical shape. A fiber layer having a uniform thickness is formed on the elliptical head portion. The head portion on which the fiber layer is formed includes a horizontal long axis portion 111 and a horizontal short axis portion 112.

In this specification, the term "transverse axis portion" used while referring to the head of the swap tool on which the fiber layer is formed represents the longest axis length among axes perpendicular to the longitudinal axis of the swap tool in the head of the elliptical swap tool. part, and the "transverse axis part" means the remaining axis parts other than the part representing the length of the longest axis among the vertical axes with respect to the longitudinal axis.

According to one embodiment of the present invention, the head portion on which the fiber layer is formed has an oval shape, has a horizontal long axis portion and a horizontal short axis portion, and the thickness of the head portion at the horizontal long axis portion is 3.8 -6.0 mm, and the thickness of the head in the transverse axis is 2.0-3.5 mm.

More specifically, the head portion on which the fiber layer is formed has an elliptical shape, has a horizontal long axis portion and a horizontal short axis portion, and has a thickness of the head portion at the horizontal long axis portion of 4.8-5.2 mm, The thickness of the head portion at the transverse axis portion is 2.8-3.2 mm.

According to one embodiment of the present invention, the fibers of the fiber layer may have a Dtex value of 3.00-3.50, more specifically, a Dtex value of 3.30-3.35.

The above-described thickness and Dtex value of the head portion on which the fibrous layer is formed allow the swab tool to have a suitable absorption capacity and to have a suitable release function when immersed in a specimen transport medium.

The fibers of the fibrous layer are materials having water affinity properties, and are specifically selected from the group consisting of polyester, polyamide and cotton.

According to one embodiment of the present invention, the head unit 11 collects a sample of 30 µl or more. Specifically, the amount of the sample to be collected is 30-1500 μl, 30-1200 μl, 30-1000 μl, 30-800 μl, 30-500 μl, 30-300 μl, 30-100 μl, 50-1500 μl, 50 μl. -1200 μl, 50-1000 μl, 50-800 μl, 50-500 μl, 50-300 μl, 50-100 μl, 100-1500 μl, 100-1200 μl, 100-1000 μl, 100-800 μl, 100 -500 μl, 100-300 μl, 200-1500 μl, 200-1200 μl, 200-1000 μl, 200-800 μl, 200-500 μl, 200-300 μl, 500-1500 μl, 500-1200 μl, 500 -1000 μl, 500-800 μl, 800-1500 μl, 800-1200 μl, 800-1000 μl, 1000-1500 μl, or 1000-1200 μl.

According to one embodiment of the present invention, the gripping portion 13 is easily cut in the cutting groove 12 .

Contamination of the sample through the gripper can be prevented by collecting a sample using the sample collection swab tool, impregnating the container containing the sample transport medium with the swab tool, and then cutting and removing the gripper from the cutting groove.

More specifically, the swap tool is easily cut in the cutting groove by using the end of the head part 11 as an action point, the cutting groove 12 as a support point, and the end of the gripping part 13 as a force point. The gripper can be easily removed by impregnating the container containing the sample transport medium with the swab tool 100 of the present invention and then positioning the cut groove on the outer circumferential surface of the container to use the cut groove as a fulcrum.

More specifically, the length between the fulcrum point and the fulcrum point and the length between the fulcrum point and the fulcrum point are 1.4-3.0:1. By adopting a sample collection swab tool with a specific length ratio based on the support point, that is, 1.4-3.0: 1, the sampled swab tool is impregnated into the container containing the sample transport medium, and then the holding part is cut with minimal force. can be removed by

Specifically, the length between the fulcrum point and the fulcrum point and the length between the fulcrum point and the action point are 1.4-2.9: 1, 1.4-2.8: 1, 1.4-2.6: 1, 1.4-2.5: 1, 1.4-2.4: 1, 1.4 -2.2:1, 1.4-2.0:1, 1.4-1.9:1, 1.6-2.9:1, 1.6-2.8:1, 1.6-2.6:1, 1.6-2.5:1, 1.6-2.4:1, 1.6-2.2 :1, 1.6-2.0:1, 1.6-1.9:1, 1.8-2.9:1, 1.8-2.8:1, 1.8-2.6:1, 1.8-2.5:1, 1.8-2.4:1, 1.8-2.2:1 , 1.8-2.0:1, 1.8-1.9:1, 1.9-2.9:1, 1.9-2.8:1, 1.9-2.6:1, 1.9-2.5:1, 1.9-2.4:1, 1.9-2.2:1, 1.9 -2.0:1, 2.0-2.9:1, 2.0-2.8:1, 2.0-2.6:1, 2.0-2.5:1, 2.0-2.4:1, 2.0-2.2:1, 2.2-2.9:1, 2.2-2.8 :1, 2.2-2.6:1, 2.2-2.5:1, 2.2-2.4:1, 2.4-2.9:1, 2.4-2.8:1, 2.4-2.6:1, 2.4-2.5:1, 2.6-2.9:1 , or 2.6-2.8:1.

The support part 10 and the gripping part 13 may be made of a plastic material. For example, the support part 10 and the gripping part 13 may be made of polypropylene, polyester or polyamide (PA66 or nylon 66).

The thickness or diameter of the support part 10 and the gripping part 13 in a cross section perpendicular to its central axis may be the same as or different from each other. The thickness or diameter is 1-5 mm, specifically 1-4 mm, 1-3 mm, 1-2 mm, 2-5 mm, 2-4 mm, 2-3 mm, 3-5 mm, 3- 4 mm or 4-5 mm. The sample collection swap tool of FIG. 1C shows that the thickness or diameter of the support part and the grip part are the same.

According to another aspect (third aspect) of the present invention, there is provided a sample collection swap tool comprising:

(a) a supporting part having a cut groove formed on an outer circumferential surface of one end thereof;

(b) a head part having a fiber layer formed at the other end of the support part to collect samples; and

(c) a gripping part extending from the cut groove of the upper limb support; In addition, the support portion includes a display portion displaying a limiting length of nasal insertion at an end of the support portion adjacent to the head portion.

The sample collection swab tool of the present invention is characterized in that it induces proper insertion of the swab tool into the anterior nasal cavity for collecting an anterior nasal swab sample by including a display unit displaying a limited length of nasal insertion.

The above description of the sample collection swap tool of the first and second aspects of the present invention can be equally applied to the swap tool of the third aspect of the present invention.

According to one embodiment of the present invention, the display unit includes grooves, protrusions, color bands, or a combination thereof formed at predetermined intervals on an outer circumferential surface of the support unit.

More specifically, the color band includes a color line.

More specifically, the display unit displays the limit length of nasal insertion for each age group. In other words, the limited length of nasal insertion for each age group is displayed on the display unit.

As long as the sample collection swap tool according to the third aspect of the present invention includes a display unit, the swap tool may have various total lengths. Specifically, the overall length of the swap tool may be selected from 60-200 mm. For example, the overall length is 60-200 mm, 70-190 mm, 80-180 mm, 90-170 mm, 100-160 mm, 110-150 mm, or 120-140 mm. According to another embodiment of the present invention, the overall length of the swap tool is 90-120 mm.

As long as the sample collection swab tool according to the third aspect of the present invention includes a display unit, the ratio of the sum of the lengths of the head unit and the support unit to the length of the holding unit may vary. Specifically, the sum of the lengths of the head part and the support part and the length of the gripping part are 1.4-3.0:1.

According to one embodiment of the present invention, the specimen is a nostril specimen and/or an oral specimen.

According to another embodiment of the present invention, the sampling swap tool is a self sampling tool.

According to another embodiment of the present invention, the lengths of the support part, the head part and the gripping part are 3-5: 1 : 2-2.8.

According to one embodiment of the present invention, the thickness of the fiber layer formed on the head portion is 0.6-3 mm.

According to another embodiment of the present invention, the head portion on which the fiber layer is formed has an oval shape, has a horizontal long axis portion and a horizontal short axis portion, and the thickness of the head portion at the horizontal long axis portion is 3.8 -6.0 mm, and the thickness of the head in the transverse axis is 2.0-3.5 mm.

According to another embodiment of the present invention, the fibers of the fiber layer have a Dtex value of 3.00-3.50.

According to one embodiment of the present invention, the fibers of the fibrous layer are selected from the group consisting of polyester, polyamide and cotton.

According to another embodiment of the present invention, the head unit collects a sample of 30 µl or more.

According to another embodiment of the present invention, the gripping portion is easily cut in the cutting groove.

More specifically, the swap tool is easily cut in the cutting groove by using the end of the head part as an action point, the cutting groove as a support point, and the end of the gripping part as a force point.

According to another aspect of the present invention, there is provided a sampling kit for detecting respiratory pathogens comprising:

(a) a sample collection swab tool of the present invention described above and (b) a container containing a sample transport medium.

Since the sample collection kit of the present invention includes the above-described sample collection swap tool of the present invention, descriptions of common contents between the two are omitted in order to avoid excessive complexity in the present specification.

According to one embodiment of the present invention, the sample collection kit is a self-sampling kit.

According to one embodiment of the present invention, the specimen transport medium is a cell preservation medium or a cell inactivation medium.

According to one embodiment of the present invention, the cell preservation medium is a saline-based solution or a balanced salt solution-based medium.

According to one embodiment of the present invention, the saline-based solution is phosphate buffered saline (PBS) or normal saline.

According to one embodiment of the present invention, the cell inactivation medium contains (i) a chaotropic agent. More specifically, the cell inactivation medium is (ii) detergent; (iii) chelators; (iv) a buffer; and (v) a reducing agent.

According to one embodiment of the present invention, when the sample transport medium is impregnated with the sample collected by the sample collection swap tool, it is used for a nucleic acid amplification reaction without a nucleic acid separation process.

Optionally, the sample transport medium further contains a pH indicator (eg, phenol red, bromocresol purple, and bromothymol blue).

According to another aspect of the present invention, the present invention provides a kit for detection of a respiratory pathogen comprising:

(a) the above-described sampling kit of the present invention; and

(b) a real-time nucleic acid amplification reaction composition comprising (i) a primer for amplifying the nucleic acid molecule of the respiratory pathogen, (ii) a probe hybridized to the nucleic acid molecule of the respiratory pathogen, and (iii) an enzyme including DNA polymerase .

Since the kit for detecting respiratory pathogens of the present invention includes the above-described sample collection kit of the present invention, descriptions of common contents between the two are omitted in order to avoid excessive complexity in the present specification.

According to one embodiment of the present invention, the enzymes include hot start Taq polymerase and reverse transcriptase.

According to one embodiment of the present invention, the nucleic acid amplification reaction composition further includes primers for amplifying endogenous IC and exogenous IC.

According to one embodiment of the present invention, the endogenous IC is RNase P gene, betaglobin gene, GAPDH gene, beta actin gene, PPIA gene, RPS9 gene, RPS15A gene or UXT gene, and the exogenous IC is bacteriophage MS2 or amourd (armoured) RNA.

According to one embodiment of the present invention, the respiratory pathogen is a respiratory virus and/or a respiratory bacterium. More specifically, the respiratory virus is influenza virus, respiratory syncytial virus (RSV), adenovirus, enterovirus, parainfluenza virus (PIV), metapneumovirus (MPV), bocavirus, rhinovirus and / or coronavirus. More specifically, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

According to one embodiment of the present invention, the nucleic acid molecule of the respiratory pathogen is at least one of the RdRP gene, S gene and N gene of SARS-CoV-2.

According to another aspect of the present invention, the present invention provides a sampling method for detecting respiratory pathogens comprising the steps of:

(a) obtaining a nasal swab sample and an oral swab sample using the above-described sample collection kit of the present invention; and

(b) impregnating the nasal swab sample and the oral swab sample into a transport medium in one container.

The sample collection method of the present invention uses the above-described sample collection kit of the present invention, and descriptions of common details between the two are omitted in order to avoid excessive complexity in the present specification.

According to another aspect of the present invention, the present invention provides a method for detecting respiratory pathogens comprising the following steps:

(a) Nucleic acid amplification reaction using a transport medium and a nucleic acid amplification reaction composition into which the whole nasal swab specimen and the oral swab specimen collected by the above-described specimen collection swab tool of the present invention are dipped together carrying out; and

(b) analyzing the result of the nucleic acid amplification reaction to determine the presence or absence of the respiratory pathogen.

The method for detecting respiratory pathogens of the present invention uses the above-described sample collection swap tool of the present invention, and descriptions of common contents between the two are omitted in order to avoid excessive complexity in the present specification.

The detection method of the present invention is described according to each step as follows:

Step (a): Nucleic acid amplification reaction using nostril swab specimen and oral swab specimen

First, using a transport medium and a nucleic acid amplification reaction composition in which the nostril swab sample and oral swab sample collected by the above-described sample collection swab tool of the present invention are dipped together, A nucleic acid amplification reaction is carried out.

The specimen used in the present invention is a nasal swab specimen and an oral swab specimen.

As used herein, the term "anterior nasal swab specimen" refers to a swab specimen in which a biopsy in the anterior nasal cavity obtained by applying a swab tool to the anterior nasal cavity is adsorbed, and is a specimen distinct from a nasopharyngeal swab specimen. For example, referring to FIG. 3A , a swab instrument is inserted into the nasal cavity (eg, inserted 2-3 cm), and a biopsy is taken from the inner portion of the nasal cavity while gently rotating the swab for 10-15 seconds. . The term “nasal swab specimen” refers to a “nasal swab specimen” or “a sample taken from the anterior nasal cavity” insofar as the nasal swab sample is obtained by applying a swab tool to the anterior nasal cavity as described above. Used interchangeably with “swab specimen collected from anterior nasal cavities.”

According to one embodiment of the present invention, an anterior nasal swab specimen is obtained by applying one swab tool to both anterior nasal cavities.

The oral swab specimen used in the present invention may include a specimen obtained by applying a swab tool to the oral cavity.

As used herein, the term "oral swab specimen" refers to a specimen obtained by collecting saliva and/or epithelial cells of the oral wall by applying a swab tool to the oral cavity.

According to one embodiment of the present invention, the oral swab sample is obtained by applying the swab tool to the lingual part, sublingual part or oral wall.

For example, the swab tool is applied to the surface of the tongue so that the fibrous layer of the swab tool is sufficiently absorbed, or the saliva is collected from the tip of the tongue and then the fibrous layer of the swab tool is sufficiently absorbed (see Fig. 3b), An oral swab sample can be obtained by rubbing the swab tool on the oral wall so that saliva is sufficiently absorbed into the fibrous layer and epithelial cells are adsorbed to the fibrous layer.

Alternatively, an oral swab sample may be obtained by sufficiently absorbing saliva while swabbing the sublingual portion (see FIG. 3B).

According to one embodiment of the present invention, the oral swab specimen is obtained by applying the swab tool to the sublingual region.

According to one embodiment of the present invention, the oral swab sample may be obtained by applying a swab tool to the inside of the cheek, gingiva and/or palate.

As used herein, the term “oral swab sample” refers to an oral swab sample or an oral saliva swab sample, as long as it is obtained by applying a swab tool to an appropriate part as described above to mainly collect saliva, are used interchangeably with each other.

According to one embodiment of the present invention, the nasal swab specimen and the oral swab specimen are obtained by applying the two swab tools to the anterior nasal cavity and oral cavity, respectively. Alternatively, anteronasal swabs and oral swabs are obtained by applying one swab tool to the anterior nasal cavity and then to the oral cavity.

According to one embodiment of the present invention, the nasal swab specimen and the oral swab specimen are obtained through self-sampling. The nasal swab specimen and the oral swab specimen are specimens that can be easily obtained by ordinary people without the involvement of experts, and therefore, effective specimens necessary for molecular diagnosis can be obtained even by self-sampling.

Since the sample collection swap tool for collecting the nasal swab sample and the oral swab sample uses the above-described sample collection swap tool of the present invention, the description of the common content between the two is omitted in order to avoid excessive complexity in the present specification. do.

Dip, i.e., immerse the sampled nasal swab and oral swab in the sample transport medium in the container. By this dipping, the sample contained in the swab is released into the sample transport medium. In general, a container containing a specimen transport medium into which a swab is dipped is transported to a place where a nucleic acid amplification reaction is performed.

The description of the sample transport medium described in the method of the present invention is equally applicable to the sample transport medium included in the sample collection kit of the present invention described above.

The specimen transport medium that can be used in the present invention includes two mediums: a cell maintenance buffer and a cell inactivation buffer (i.e., cell disruption medium).

As used herein, the term “cell preservation medium” refers to a medium that maintains cells in a specimen so as not to be disrupted.

The cell preservation medium that can be used in the present invention includes any cell preservation medium commonly used in the art. According to one embodiment of the present invention, the cell preservation medium is a saline-based solution or a balanced salt solution-based medium.

More specifically, the saline-based solution used in the present invention is phosphate buffered saline (PBS) or normal saline.

The balanced salt solution-based medium used in the present invention may include a balanced salt solution commonly used in the art. For example, balanced salt solutions include Alsever's solution, Earle's balanced salt solution, Gey's balanced salt solution, Hanks' balanced salt solution, These include Puck's balanced salt solution, Ringer's balanced salt solution, Simm's balanced salt solution and Tyrode's balanced salt solution.

The balanced salt solution-based medium used in the present invention may further include other components in the above-described balanced salt solution. For example, the other components include bovine serum albumin, cysteine, sucrose and glutamic acid.

The cell inactivation medium used in the present invention, i.e., disruptive medium, disrupts a sample, such as a virus, to release nucleic acid molecules within the virus into the medium, denatures protein components, inactivates DNase and RNase, and Integrity is a medium to maintain.

More specifically, the cell inactivation medium as the specimen transport medium used in the present invention contains (i) a chaotropic agent, and more specifically (i) in addition to the chaotropic agent (ii) dietary detergent; (iii) chelators; (iv) a buffer; and (v) a reducing agent, and more particularly, in addition to (i) a chaotropic agent, (ii) a detergent; (iii) chelators; (iv) a buffer; and (v) a reducing agent.

The chaotropic agent is guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride and potassium thiocyanate, and more specifically, guanidine thiocyanate )am. Chaotropic agents open microbial cells, induce cell lysis, release DNA and RNA, and degrade nucleic acid molecules by nucleases ( degradation) is prevented.

Specifically, the detergent is sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate ( sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, polysorbate, Triton X-100 or N -Lauroyl sarcosine (N-lauroyl sarcosine).

Specifically, the chelator is ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N, N-bis (carboxy Methyl)glycine (N,N-bis(carboxymethyl)glycine), ethylenediaminetetraacetic acid, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate (ammonium citrate), ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate or lithium citrate.

Specifically, the buffer is tris (hydroxymethyl) aminomethane (tris (hydroxymethyl) aminomethane), citrate (citrate), 2- (N-morpholino) ethanesulfonic acid (2- (N-morpholino) ethanesulfonic acid ), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid), 1,3-bis (tris (hydroxymethyl) )methylamino)propane (1,3-bis(tris(hydroxymethyl)methyl amino)propane), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (4-(2-hydroxyethyl)-1- piperazine ethanesulfonic acid), 3-(N-morpholino)propanesulfonic acid, hydroxyethyl piperazine ethane sulfonic acid, bicarbonate and phosphate.

Specifically, the reducing agent is 2-mercaptoethanol, tris (2-carboxyethyl) phosphine, dithiothreitol, dimethylsulfoxide and sodium hydroxide.

In the sample transport medium used in the present invention, the chaotropic agent may be included in an amount of 5-30 parts by weight based on 100 parts by weight of the medium, the detergent is 1-15 parts by weight, the chelator is 0.1-5 parts by weight, and the buffer solution 2-10 parts by weight of silver and 0.1-3 parts by weight of the reducing agent may be included.

According to one embodiment of the present invention, the cell inactivation medium as a sample transport medium has an inactivating function by disruption of respiratory infectious pathogens and nucleic acid materials (specifically, DNA or RNA, more specifically RNA) released from the disrupted pathogens. has a stabilizing function.

Optionally, the sample transport medium further includes a pH indicator (eg, phenol red, bromocresol purple, and bromothymol blue), more specifically, phenol red. The pH indicator makes it possible to monitor whether the sample transport medium is infected with bacteria.

A nucleic acid amplification reaction is carried out using a sample transport medium. Sample transport media can be applied to nucleic acid amplification reactions in three ways:

According to the first method, the specimen transport medium is directly applied to the nucleic acid amplification reaction without any pretreatment. For example, PCR can be performed by adding a specimen transport medium to a nucleic acid amplification reaction composition according to a conventional direct PCR method.

According to the second method, after heat treatment (incubation) of the sample transport medium, the incubation result is applied to the nucleic acid amplification reaction.

Specifically, the sample transport medium in which the whole nasal swab sample and the oral swab sample are dipped together is 60°C to 100°C (more specifically, 95°C to 100°C) for 1 minute to 15 minutes before being used for a nucleic acid amplification reaction. Incubation is performed, and the nucleic acid amplification reaction is performed using the result of the incubation. In this case, the sample transport medium used in the nucleic acid amplification reaction may be diluted 2 to 10 times (more specifically, 3 to 5 times) before or after incubation and applied to the nucleic acid amplification reaction.

According to the third method, a nucleic acid separation process is applied to the specimen transport medium in which the whole nasal swab specimen and the oral swab specimen are dipped together before being used in the nucleic acid amplification reaction, and the nucleic acid molecules separated in the nucleic acid separation process are used to A nucleic acid amplification reaction is carried out.

The nucleic acid separation process may be performed, for example, according to a magnetic particle-based separation method (see US Patent Nos. 6027945 and 7517969). For example, nucleic acid molecules can be separated by sequentially using a buffer for separating nucleic acid molecules composed of a lysis buffer, a binding buffer, a washing buffer, and an elution buffer. More specifically, the sample transport medium is treated with a lysis buffer containing a chaotropic agent (e.g., guanidine thiocyanate) and a detergent (e.g., Tween-20) to disrupt viruses or bacteria in the sample, and magnetic particles are added to the disruption product. Nucleic acid molecules bound to the surface of the magnetic particles are separated by treatment with a binding buffer containing , washing the magnetic particles with a washing buffer containing ethanol, and then treatment with an elution buffer to separate the nucleic acid molecules bound to the surfaces of the magnetic particles.

For the nucleic acid amplification reaction, primers and probes that hybridize to the target may be used.

The probe or primer used in the present invention has a sequence complementary to the target nucleotide sequence. As used herein, the term "complementary" means that it has sufficient complementarity to selectively hybridize to the above-mentioned nucleotide sequence under certain specific hybridization or annealing conditions. Therefore, the term "complementary" has a different meaning from the term perfectly complementary, and the primers or probes of the present invention have one or more mismatches ( mismatch) nucleotide sequence.

As used herein, the term "primer" refers to a single agent capable of acting as the starting point of template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. -means a stranded oligonucleotide. The suitable length of a primer varies depending on various factors, such as temperature and use of the primer, but is typically 15-40 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.

The sequence of the primer does not have to have a sequence completely complementary to a part of the sequence of the template, and it is sufficient to have sufficient complementarity within the range of hybridizing with the template to perform the specific function of the primer. Therefore, the primer in the present invention does not have to have a sequence perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within the range in which it can hybridize to this gene sequence and act as a primer. The design of such primers can be easily performed by those skilled in the art by referring to the above-described nucleotide sequences, and can be performed using, for example, a primer design program (eg, PRIMER 3 program).

As used herein, the term “probe” refers to a natural or modified monomer or linear oligomer of linkages, including deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleotide sequence, and naturally existing or artificially synthesized. The probe of the present invention is preferably single-stranded and is an oligodeoxyribonucleotide.

As used herein, the term “amplification” refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), the method of Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR) ( 17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self-maintaining sequence replication (self sustained sequence replication, WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (consensus sequence primed polymerase chain reaction) CP-PCR), U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence-based amplification based amplification (NASBA), U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP), but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09/854,317.

PCR is the most well-known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For details on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

Most respiratory pathogens have RNA as their genome. In this case, a gene amplification reaction is performed using RNA in the sample as a template and primers binding to RNA or cDNA. cDNA is synthesized from the isolated RNA, and the cDNA is amplified. cDNA can be readily synthesized using reverse transcriptase (PNAS USA, 85:8998 (1988); Libert F, et al., Science , 244:569 (1989); and Sambrook, J. et al. , Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). Subsequently, the synthesized cDNA is amplified through a gene amplification reaction.

The primer used in the present invention is hybridized or annealed to one site of the template to form a double-stranded structure. Conditions of nucleic acid hybridization suitable for forming such a double-stranded structure are described in Joseph Sambrook, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used for the amplification of the present invention, including the “Klenow” fragment of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which include Thermos aquaticus (Taq), Thermos thermophilus (Tth), Thermos filipformis ( Thermus filiformis ), Thermis flavus, Thermococcus literalis , and Pyrococcus furiosus (Pfu).

When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with components necessary for the reaction in excess. An excess of components required for the amplification reaction means an amount to the extent that the amplification reaction is not substantially limited by the concentration of the components. It is required to provide cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture in such a way that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

According to one embodiment of the present invention, the nucleic acid amplification reaction used in the present invention is a real-time nucleic acid amplification reaction.

The real-time nucleic acid amplification reaction may be performed using a non-specific fluorescent dye that is non-specifically intercalated with a duplex, which is an amplicon of a target nucleic acid sequence.

In addition, the real-time nucleic acid amplification reaction may use a probe that specifically hybridizes to a target nucleic acid sequence. For example, the method is a molecular beacon method (Tyagi et al, Nature Biotechnology v.14 MARCH 1996) using a dual-labeled probe that forms a hairpin structure, as a donor or acceptor. Hybridization probe method using two single labeled probes (Bernad et al, 147-148 Clin Chem 2000; 46) and Lux method using single labeled oligonucleotides (U.S. Patent No. 7,537,886), dual labeled It includes, but is not limited to, the TaqMan method (U.S. Patent Nos. 5,210,015 and 5,538,848), which utilizes hybridization of probes as well as cleavage of dual-labeled probes by 5'-nuclease activity of DNA polymerase.

In addition, a real-time nucleic acid amplification reaction can be performed using a dimer formed depending on the presence of a target nucleic acid sequence. The dimer formed depending on the presence of the target nucleic acid sequence is not an amplification product of the target sequence formed by the amplification reaction itself, but is a dimer whose amount increases in proportion to the amplification of the target nucleic acid sequence. Dimers formed depending on the presence of a target nucleic acid sequence can be obtained by various methods, for example, by the PTO Cleavage and Extension (PTOCE) method disclosed in WO 2012/096523, the above patent documents being incorporated herein by reference. inserted into

According to one embodiment of the present invention, the nucleic acid amplification reaction composition includes a primer, a probe and an enzyme. The nucleic acid amplification reaction composition includes a buffer, dNTP, KCl and MgCl ₂ . For example, the nucleic acid amplification reaction composition may include 40 mM Tris·Cl (pH 8.8), 100 mM KCl, 4 mM MgCl ₂ and 400 μM dNTP. Enzymes included in the nucleic acid amplification reaction composition of the present invention include Taq DNA polymerase (specifically, hot start Taq DNA polymerase) and reverse transcriptase, and more specifically, hot start Taq DNA polymerase, reverse transcriptase, UDG (Uracil DNA glycosylase) and RI (RNase inhibitor). The hot start Taq DNA polymerase is chemically modified hot start Taq DNA polymerase (Reference: Paul N, et al., Hot start PCR. Methods in Molecular Biology. Humana Press. 630:301-18 (2010)), antibody- based hot start Taq DNA polymerase (Paul N, et al., Hot start PCR. Methods in Molecular Biology. Humana Press. 630:301-18 (2010)) and aptamer-based hot start Taq DNA polymerase (Birch DE , et al., Simplified hot start PCR. Nature 381:445-446 (1996)). The reverse transcriptase used in the present invention is a reverse transcriptase commonly used in the art, specifically an MMLV-based thermostable reverse transcriptase.

According to one embodiment of the present invention, the nucleic acid amplification reaction composition includes primers, probes and enzymes (hot start Taq DNA polymerase, MMLV-based thermostable reverse transcriptase and UDG).

In general, an internal control (IC) is used in a nucleic acid amplification reaction to determine the validity of the nucleic acid amplification reaction. In the present invention, the sampling effectiveness of the nasal swab specimen and oral swab specimen as well as the nucleic acid amplification reaction can be determined using two ICs.

According to one embodiment of the present invention, the nucleic acid amplification reaction composition includes primers for amplifying nucleic acid molecules of the respiratory pathogen, endogenous IC and exogenous IC, wherein the endogenous IC is the former. The sampling validity of nasal swabs and oral swabs is determined and the exogenous IC is used to determine the validity of the nucleic acid amplification reaction.

According to one embodiment of the present invention, the endogenous IC is RNase P gene, betaglobin gene, GAPDH gene, beta actin gene, PPIA gene, RPS9 gene, RPS15A gene or UXT gene, and the exogenous IC is bacteriophage MS2 or amourd (armoured) RNA.

Step (b): Determination of Respiratory Pathogen Presence

Finally, the presence or absence of the respiratory pathogen is determined by analyzing the result of the nucleic acid amplification reaction.

For example, a signal of a real-time nucleic acid amplification reaction is detected in real time, and when a signal equal to or higher than a specific threshold value is observed, it is determined that a respiratory pathogen is present.

According to one embodiment of the present invention, the respiratory pathogens detected by the present invention are respiratory viruses and/or respiratory bacteria.

For example, the present invention relates to influenza virus (eg, influenza A virus and influenza B virus), respiratory syncytial virus (RSV) (eg RSV A and RSV B), adenovirus, enterovirus, parainfluenza virus (PIV) (eg , PIV 1, PIV 2, PIV 3 and PIV 4), metapneumovirus (MPV), bocavirus, rhinovirus, coronavirus (eg CoV NL63, CoV 229E, CoV OC43, CoV HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2), Mycoplasma pneumoniae , Chlamydophila pneumoniae , Legionella pneumophila , Haemophilus influenzae , Streptococcus pneumoniae , Bordetella pertussis and/or Bordetella parapertussis infection.

According to one embodiment of the present invention, the present invention is an influenza virus, RSV (respiratory syncytial virus), adenovirus, enterovirus, PIV (parainfluenza virus), MPV (metapneumovirus), bocavirus, rhinovirus and / or coronavirus infection by respiratory viruses, including

More specifically, the method of the present invention determines infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

According to one embodiment of the present invention, the nucleic acid amplification reaction composition is used to amplify at least one, more specifically at least two, and more specifically three genes among the RdRP gene, S gene and N gene of SARS-CoV-2. It is determined that the pathogen of SARS-CoV-2 is present when at least one of the RdRP gene, the S gene, and the N gene is measured as positive.

According to one embodiment of the present invention, the present invention is performed by a real-time nucleic acid amplification reaction that exhibits a LoD (Limit of Detection) value of 100-500 RNA copies/reaction.

The features and advantages of the present invention are summarized as follows:

(a) In the case of using a swab tool used as a conventional nasopharyngeal swab tool as an anterior nasal swab tool and/or an oral swab tool, the overall length of the swab tool is long and the length of the gripping part is the length of the head part and the support part. There is no difference from the sum of , making it difficult to accurately sample in the anterior nasal cavity and/or oral cavity, and even trying to insert it into the posterior wall of the nasopharynx and oropharynx. ), there was a problem in that it was difficult to collect the required amount of sample.

(b) However, the present invention reduces the overall length of the sample collection swap tool to a specific length, or sets the ratio of the sum of the lengths of the head portion and the support portion to the length of the gripping portion constituting the sample collection swap tool to a specific length. value, to accurately and easily take an anterior nasal swab sample and/or an oral swab sample from the sampling site (specifically, the anterior nasal cavity and/or oral cavity) and collect a sufficient amount of sample to detect a respiratory pathogen. can

(c) In addition, the present invention can be used not only for sampling by experts but also for self-sampling by non-experts, and can detect respiratory pathogens from collected whole nasal swab samples and oral swab samples. there is.

1A shows an example of applying a swab tool used as a conventional nasopharyngeal swab tool to a nasal swab tool and an oral swab tool.
1B to D show a sampling swab tool used to collect an anterior nasal swab specimen and/or an oral swab specimen according to one embodiment of the present invention. The sample collection swap tool includes a display unit on which a limiting length for nasal insertion is displayed at an end of the support unit. In the sample collection swap tool shown in FIG. 1C , the thickness of the support part and the grip part are the same.
2 shows a head portion of a sample collection swab tool according to an embodiment of the present invention.
3A exemplarily shows how to collect a sample using a nasal swab tool.
3B exemplarily shows how to collect a sample using an oral swab tool.

Hereinafter, the present invention will be described in more detail through examples. These embodiments are only for explaining the present invention in more detail, and it is to those skilled in the art that the scope of the present invention is not limited by these embodiments according to the gist of the present invention. It will be self-evident.

Example

Comparative Example 1: Detecting SARS-CoV-2 by taking a nasal swab sample and an oral swab sample

Using the sample collection swap tool (total length: 157 mm, head length: 15 mm, support portion length: 66 mm, gripping portion length: 76 mm) shown in FIG. collected. The nasal swab tool and oral swab tool used were the same, and included an elliptical head on which a fibrous layer was formed, and the thickness of the head at the transverse axis was 5.0 mm, and the head at the transverse axis was 3.0 mm. . The fiber layer of the swab is formed by coating 3.30-3.35 Dtex polyester fiber with flocking technology.

The nasal cavity swab tool was inserted 2 cm into the nasal cavity and gently rotated for 20 seconds while swabbing was performed to obtain a nasal cavity swab specimen. Swabing was performed in one anterior nasal cavity, followed by swabbing in the other anterior nasal cavity. Subsequently, an oral swab sample was obtained by applying an oral swab tool to the sublingual portion and swabbing so that saliva was sufficiently absorbed in the fibrous layer of the swab.

Then, the nasal swab specimen and the oral swab specimen were impregnated in a balanced salt solution-based medium, CTM (Clinical Virus Transport Medium) (Noble Bio Company) or UTM (Copan Company) specimen transport medium. Subsequently, target nucleic acids to be added to the RT-PCR reaction were obtained by heat-treating the specimen transport medium or through a nucleic acid isolation process.

To obtain a target nucleic acid by heat treatment, a sample transport medium impregnated with a nasal swab sample and an oral swab sample was diluted 3-fold and incubated at 98° C. for 3 minutes to obtain a target nucleic acid (hereinafter referred to as “crude nucleic acid”). called).

Nucleic acid isolation was performed using Seegene's STARMag 96X4 Universal Cartridge Kit (Cat. No. 744300.4.UC384, Seegene Inc.) and automatic nucleic acid extraction equipment Microlab NIMBUS (Cat. No. 65415-02, Hamilton). Nucleic acid separation was carried out according to the manufacturer of the nucleic acid separation reagent and the device operation manual. Nucleic acid isolation was performed using 300 μl of the specimen transport medium impregnated with the whole nasal swab specimen and the oral swab specimen (hereinafter referred to as "isolated nucleic acid").

The RT-PCR reaction using the crude nucleic acid or the isolated nucleic acid was performed using Allplex ^TM SARS-CoV-2 Assay (Seegene), a multi-one step RT-PCR product. Allplex ^TM SARS-CoV-2 Assay product is a one-step RT-PCR product for detecting Sarbecovirus and SARS-CoV-2 target gene E gene, SARS-CoV-2 target gene RdRP gene, S gene and N gene.

5 μl of crude or isolated nucleic acid, 5 μl of enzyme (including chemically modified hot start Taq polymerase, RTase, UDG and RI), 5 μl of RNase-free Water and 5 μl of SARS2 MOM (Oligo mix) to a final volume of 20 μl A reaction mixture for RT-PCR reaction was prepared.

The tubes each containing the prepared reaction mixture were placed in a real-time thermocycler (CFX96, Bio-Rad), reacted at 50°C for 20 minutes, then denatured at 95°C for 15 minutes, 95°C for 10 seconds, 60°C. 15 seconds at 72 ° C. and 10 seconds were repeated for 45 cycles. The above experiment was repeated twice to obtain an average Ct value.

As a result of the experiment, a positive signal for SARS-CoV-2 was obtained when using isolated nucleic acids obtained from nasal swab samples and oral swab samples. In addition, even when crude nucleic acids were used from nasal swab samples and oral swab samples, positive signals for SARS-CoV-2 could be obtained, but compared to isolated nucleic acids, the Ct value was delayed by 1-3, which was The sensitivity is slightly reduced by 3-6 times.

Example 1: Detecting SARS-CoV-2 by taking an anterior nasal swab sample and an oral swab sample

The whole nasal swab sample and the oral swab were performed in the same manner as in Comparative Example 1, except that the nasal swab sample and the oral swab sample were collected using the sample collection swap tool shown in B to D of FIG. 1 as the sample collection swap tool. A sample was taken to detect SARS-CoV-2. The sample collection swap tools of FIG. 1 B and C have a total length of 114 mm, a head length of 15 mm, a support portion length of 66 mm, and a holding portion length of 33 mm, and the sample collection swap tool of FIG. 1 D has an overall length of 95 mm. , with a head length of 15 mm, a support section length of 47 mm and a grip section length of 33 mm.

As a result of the experiment, when using the sample collection swap tool shown in B to D of FIG. 1, an equivalent positive rate for SARS-CoV-2 was shown compared to the case where the sample collection swap tool shown in A of FIG. 1 was used. However, it was not easy to insert the sample collection swab tool of FIG. 1 A 2 cm into the anterior nasal cavity, and there were problems such as stabbing the nasal cavity wall or inserting it deeply through the nasal cavity formed under the lower nasal irrigation. Compared to D's sample collection swap tool, it took 3-4 times more time to collect samples.

Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (33)

A sampling swap tool that includes:
(a) a supporting part having a cut groove formed on an outer circumferential surface of one end thereof;
(b) a head part having a fiber layer formed at the other end of the support part to collect samples; and
(c) a gripping part extending from the cut groove of the upper limb support; And, the total length of the swap tool is 90-120 mm.
A sampling swap tool that includes:
(a) a supporting part having a cut groove formed on an outer circumferential surface of one end thereof;
(b) a head part having a fiber layer formed at the other end of the support part to collect samples; and
(c) a gripping part extending from the cut groove of the upper limb support; And, the sum of the lengths of the head part and the support part and the length of the gripping part are 1.4-3.0:1.
A sampling swap tool that includes:
(a) a supporting part having a cut groove formed on an outer circumferential surface of one end thereof;
(b) a head part having a fiber layer formed at the other end of the support part to collect samples; and
(c) a gripping part extending from the cut groove of the upper limb support; In addition, the support portion includes a display portion displaying a limiting length of nasal insertion at an end of the support portion adjacent to the head portion.
4. The swab tool according to any one of claims 1 to 3, wherein the sample is a nostril sample and/or an oral cavity sample.
4. The sampling swap tool according to any one of claims 1 to 3, wherein the sampling swap tool is a self sampling tool.
The sample collection swab tool according to any one of claims 1 to 3, wherein the lengths of the support part, the head part, and the grip part are 3-5: 1: 2-2.8.
The swab tool for collecting samples according to claim 1 or 2, wherein the support unit includes a display unit displaying a limiting length for nasal insertion at an end of the support unit adjacent to the head unit.
The sample collection swab tool according to claim 3 or 7, wherein the display unit includes grooves, protrusions, color bands, or a combination thereof formed at predetermined intervals on the outer circumferential surface of the support unit.
The swab tool for collecting specimens according to claim 3 or 7, wherein the display unit displays the limit length of nasal insertion for each age group.
The swab tool for collecting samples according to any one of claims 1 to 3, wherein the fiber layer formed on the head part has a thickness of 0.6-3 mm.
The method according to any one of claims 1 to 3, wherein the head portion on which the fiber layer is formed has an elliptical shape and has a horizontal long axis portion and a horizontal short axis portion. The thickness of the head part is 3.8-6.0 mm, and the thickness of the head part in the transverse axis is 2.0-3.5 mm.
The sampling swab tool according to any one of claims 1 to 3, wherein the fibers of the fiber layer have a Dtex value of 3.00-3.50.
4. The swab tool according to any one of claims 1 to 3, wherein the fibers of the fiber layer are selected from the group consisting of polyester, polyamide and cotton.
The sample collection swap tool according to any one of claims 1 to 3, wherein the head unit collects a sample of 30 µl or more.
The swab tool for collecting samples according to any one of claims 1 to 3, wherein the holding part is easily cut from the cutting groove.
16. The swab tool according to claim 15, wherein the swab tool is easily cut at the cutting groove by using the end of the head part as an action point, the cutting groove as a support point, and the end of the gripping part as a force point.
Sampling kits for detection of respiratory pathogens, including:
(a) a sample collection swab tool according to any one of claims 1 to 3; and (b) a container containing a sample transport medium.
18. The kit according to claim 17, wherein the specimen transport medium is a cell preservation medium or a cell inactivation medium.
19. The kit according to claim 18, wherein the cell preservation medium is a saline-based solution or a balanced salt solution-based medium.
The kit according to claim 19, wherein the saline-based solution is phosphate buffered saline (PBS) or normal saline.
19. The kit according to claim 18, wherein the cell inactivation medium comprises (i) a chaotropic agent.
22. The method of claim 21, wherein the cell inactivation medium is (ii) detergent; (iii) chelators; (iv) a buffer; And (v) a kit characterized in that it further comprises at least one component selected from the group consisting of a reducing agent.
The kit according to claim 17, wherein the sample transport medium is used for a nucleic acid amplification reaction without a nucleic acid separation process when the sample collected by the sample collection swap tool is impregnated therein.
18. The kit according to claim 17, wherein the respiratory pathogen is a respiratory virus and/or a respiratory bacterium.
25. The method of claim 24, wherein the respiratory virus is an influenza virus, RSV (respiratory syncytial virus), adenovirus, enterovirus, PIV (parainfluenza virus), MPV (metapneumovirus), bocavirus, rhinovirus and / or coronavirus. kit to.
The kit according to claim 25, wherein the coronavirus is SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).
A sampling method for detecting respiratory pathogens comprising the following steps:
(a) obtaining a nasal swab sample and an oral swab sample using the sample collection kit of claim 17; and
(b) impregnating the nasal swab sample and the oral swab sample into a transport medium in one container.
28. The method of claim 27, wherein the nostril swab specimen and the oral swab specimen are obtained through autologous sampling.
28. The method of claim 27, wherein the anterior nasal swab specimen is obtained by applying a single swab tool to both anterior nasal cavities.
28. The method of claim 27, wherein the oral swab sample is obtained by applying the swab tool to a sublingual part or oral cavity wall.
31. The method of claim 30, wherein the oral swab sample is obtained by applying a swab tool to the sublingual part.
28. The method of claim 27, wherein the nasal swab specimen and the oral swab specimen are obtained by applying the two swab instruments respectively to the anterior nasal cavity and oral cavity.
28. The method of claim 27, wherein the anterior nasal swab and oral swab are obtained by applying one swab to the anterior nasal cavity and then to the oral cavity.