KR102330620B1 - Method of providing information for diagnosis and treat monitiring of Sarcoptes scabiei infection - Google Patents

Abstract

Provided is a method for providing information for diagnosis and treatment monitoring of sarcoptes scabiei infection using dual real time qPCR. According to the present invention, sarcoptes scabiei infection can be efficiently confirmed based on improved sensitivity and accuracy.

Description

Method of providing information for diagnosis and treat monitoring of Sarcoptes scabiei infection

The present invention relates to a method for providing information for diagnosis and treatment monitoring of scabies mite infection, and more particularly, to a method for providing information for diagnosis and treatment monitoring of scabies mite infection using dual real-time qPCR.

Scabies is a highly contagious infection caused by skin parasitism of the scabies mite (Sarcoptes scabiei). Scabies is a disease that is generally prevalent in low- or middle-income countries, but its incidence in senior care facilities in high-income countries is increasing. In Korea, the incidence of scabies has decreased significantly since the end of 1980, but the incidence of scabies has recently increased in hospitals and nursing homes. On the other hand, since scabies can have specific clinical features in certain cases, including immunocompromised patients, infants or the elderly, and crystalline scabies, if most doctors rely on clinical symptoms when diagnosing scabies, scabies can be treated as other skin diseases. It can be difficult to distinguish from In addition, since scabies is highly contagious, if diagnosis is delayed, it can be transmitted to family members, and in the case of hospitals, temporary hospital closures can occur because scabies can be transmitted to other patients, caregivers, and medical staff.

Because the scabies mite is very small (approximately 200 to 450 μm in size), it cannot be observed with the naked eye, so the scabies infection test is usually by scraping the patient's skin with mineral oil, transferring it to a slide glass, and then under a microscope. This is done by identifying eggs. However, since the reliability of the test is 0.46 (95% confidence interval of 0.31 to 0.62, which is very low, there is a problem that scabies mite infection cannot be excluded even if the microscopic examination is negatively diagnosed (Arch Dermatol 2011; 147:468-73) ).

Dermoscopy is generally useful as a tool for differential diagnosis of benign pigmented lesions such as naevi, seborrhoeic keratosis, or melanoma, and is effective and effective in diagnosing scabies. It is known to be convenient to use. Dermoscopy-guided skin scraping with microscopic examination (DSGSS-ME) can be used to diagnose scabies as well as evaluate treatment efficacy. However, the method using dermoscopy is not accepted as a general method for diagnosing scabies because it requires long training. Meanwhile, although several conventional PCRs have been introduced as alternative diagnostic tools, it has not yet been studied whether dual real-time quantitative PCR as well as scabies diagnosis can be used.

Korean Patent Laid-Open Patent 10-2010-0035644 Korean Patent Publication No. 10-2007-0074626

Therefore, the present invention is to solve various drawbacks and problems caused by the prior art in consideration of the above circumstances. Diagnosis and treatment monitoring of scabies mite infection that can be diagnosed and monitored after treatment at the same time The purpose is to provide a method of providing information for

In addition, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention in order to achieve the above object, extracting a DNA sample from a biological sample isolated from an individual, designing primers and probes, amplifying by a double PCR reaction, and Provided is a method for providing information for diagnosis and treatment monitoring of scabies mite infection, comprising the step of performing dual real-time qPCR.

At this time, according to another embodiment of the present invention, when the cox1 gene is detected as a result of amplification by the double PCR reaction, it may be determined that the infection is caused by scabies mite.

In addition, according to another embodiment of the present invention, the cox1 gene may be derived from human scabies mite (Sarcoptes scabiei var hominis).

In addition, according to another embodiment of the present invention, the double real-time qPCR may be to measure the plasmid DNA concentration after inserting a sample amplified by double PCR into a plasmid vector, proliferating and purifying it.

In addition, according to another embodiment of the present invention, it may be to monitor whether the scabies mite treatment by measuring the change in the concentration of the plasmid DNA.

In addition, according to another embodiment of the present invention, the primer may be a primer for detecting a cytochrome c oxidase subunit 1 (cox1) gene derived from Sarcoptes scabiei.

In addition, according to another embodiment of the present invention, the cox1 gene of the primer may be derived from a human scabies mite (Sarcoptes scabiei var hominis).

In addition, according to another embodiment of the present invention, a kit for diagnosing scabies mite infection using the above-described information providing method is provided.

Also, according to another embodiment of the present invention, the kit may be a nested PCR kit.

Also, according to another embodiment of the present invention, the kit may be a dual real-time qPCR kit.

Meanwhile, according to another embodiment of the present invention, there is provided a composition for diagnosing scabies, including the primer and the probe of the information providing method described above.

In addition, according to another embodiment of the present invention, a primer for diagnosing scabies mite infection applied to the above-described information providing method is provided.

The method of providing information for diagnosis and treatment monitoring of scabies mite infection of the present invention uses dual real-time qPCR, which is easy and improves sensitivity and accuracy, so that it is possible to check whether scabies mite infection with excellent efficiency and at the same time whether to treat There are benefits to monitoring.

In addition, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a graph showing the change in parasitic load during the follow-up period after the start of treatment.
Figure 2 shows the sequential changes of parasitic hatching and DS test results and permethrin treatment in a 71-year-old male patient.
Figure 3 shows the qPCR test during the follow-up period.

Hereinafter, the present invention will be described in detail. Prior to this, the terms or words used in the present specification and claims are not to be construed as being limited to their ordinary or dictionary meanings, and the inventor must properly understand the concept of the term in order to best describe his invention. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined in Accordingly, the configuration described in the embodiment described in this specification is only the most preferred embodiment of the present invention and does not represent all of the technical idea of the present invention, so various equivalents and It should be understood that there may be variations.

As used herein, the term "scabies mite (Sarcoptes scabiei)" is a parasite that parasitizes in the skin of animals and the like, and is also called 'scabies' and is known to cause scabies, an infectious skin disease. Scabies mites are known as human scabies (Sarcoptes scabiei var hominis), dog scabies (Sarcoptes scabiei var canis) and pig scabies (Sarcoptes scabiei var suis).

The composition for diagnosing scabies mite infection of the present invention may be for diagnosing infection of human scabies mite (Sarcoptes scabiei var hominis), but is not limited thereto.

The term 'scabies' of the present invention is a common contagious skin disease that affects a large number of people worldwide, and can be acquired regardless of race, age, or income. Since the symptoms of scabies are similar to those of eczema, atopic dermatitis, contact dermatitis, insect bites, urticaria, and impetigo, there is a possibility of a misdiagnosis when diagnosing scabies. In addition, it is very important to receive treatment at the same time as scabies can only be felt after about a month after the scabies mite has parasitized.

In the present invention, "scabies mite infection" and "scabies outbreak" may be used interchangeably.

As used herein, the term "cox1 (cytochrome c oxidiase subunit 1) gene" is a gene encoding subunit 1 of cytochrome c oxidase, which is an oxidative phosphorylation (OXPHOS) complex IV, mitochondrial DNA is a gene present in

The cox1 gene detected by the composition for diagnosing scabies scabies infection of the present invention may be derived from scabies mites (Sarcoptes scabiei), and specifically, the cox1 gene may be derived from human scabies mites (Sarcoptes scabiei var hominis). , but is not limited thereto.

The composition for diagnosing scabies mite infection of the present invention detects the cox1 gene derived from scabies mite to diagnose scabies infection. .

As used herein, the term "primer" refers to a base sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and is used for template strand copying. A short sequence that serves as a starting point. In the present invention, the primers used for RNA amplification of the gene are prepared under suitable conditions in an appropriate buffer (for example, 4 different nucleoside triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) and suitable It may be a single-stranded oligonucleotide that can serve as a starting point for template-directed DNA synthesis under temperature, and the appropriate length of the primer may vary depending on the intended use. The primer sequence does not need to be completely complementary to the polynucleotide of the RNA of the gene or its complementary polynucleotide, and can be used as long as it is sufficiently complementary to hybridize.

The nucleotide sequence used in the present invention is construed to include a sequence exhibiting substantial identity to the sequence described in the sequence listing, in consideration of mutations having biologically equivalent activity. The term, 'substantial identity' refers to aligning the sequence of the present invention and any other sequences as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art. means a sequence that exhibits 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

As used herein, the term "diagnosis" refers to confirming the presence or characteristics of a pathological condition, and may specifically refer to determining whether scabies mite infection or scabies develops, and more specifically, the cox1 gene derived from scabies mite. It may be to detect. The kit of the present invention can analyze whether the cox1 gene derived from scabies mite is detected using the composition for diagnosing scabies mite infection. The kit may be a nested PCR kit, a nested PCR kit, an RT-PCR kit, or a DNA chip kit, and specifically, the kit may be a double PCR kit, but is not limited thereto.

The term "double PCR (nested PCR)" of the present invention is a PCR reaction in which a first primer pair is amplified and then a second primer pair is added to amplify it, and is often used to increase sensitivity or when special purpose amplification is required. As a PCR reaction method, in the present invention, double PCR was used to accurately detect the cox1 gene derived from scabies mites.

The kit may be a kit including essential elements necessary for performing double PCR. For example, the double PCR kit may include a double PCR premix, a first primer pair and a second primer pair, a DNA template, and the like, but is not limited thereto.

As another example, the kit may be a kit including essential elements necessary for performing general PCR. For example, the PCR kit may include a PCR premix, primers, DNA template, etc., but is not limited thereto.

As another example, the kit may be a kit including essential elements necessary for performing RT-PCR. For example, the RT-PCR kit may contain, in addition to each primer specific for the gene, a test tube or other suitable container, reaction buffer (Ph and magnesium concentrations vary), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs) ), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include a pair of primers specific for a gene used as a quantitative control.

As another example, the kit of the present invention may be a DNA chip kit for diagnosing scabies mite infection including essential elements necessary for performing the DNA chip. As used herein, the term "DNA chip" refers to one of the DNA microarrays that can check each base of hundreds of thousands of DNA at once. In the DNA chip kit, nucleic acid species are attached in a gridd array to a generally flat solid support plate, typically a glass surface no larger than a microscope slide, and the nucleic acids are uniformly arranged on the chip surface to form a DNA chip. It is a tool that allows multiple hybridization reactions between the nucleic acids on the chip surface and the complementary nucleic acids contained in the solution treated on the chip surface to enable massively parallel analysis.

As used herein, the term "individual" refers to any organism that is or is likely to be infected with scabies mite or any organism that develops or is likely to develop scabies, and specifically, mice, monkeys, cattle, pigs, dogs, cats , may include, without limitation, mammals including livestock, humans, and the like, and more specifically, may be humans, but is not limited thereto.

As used herein, the term "sample" refers to a material derived from an individual infected with or likely to be infected with a scabies mite or an individual who has or is likely to develop scabies, specifically, cells, blood, serum, skin, urine, or It may be hair, but the type is not limited thereto, as long as the DNA of the individual can be obtained and the existence of the cox1 gene derived from the scabies mite can be confirmed therefrom.

In an embodiment of the present invention, a skin scrape sample from a patient with scabies mite infection or abnormal symptoms was obtained, and an experiment for detecting the cox1 gene derived from scabies mite was performed using the sample. The information providing method for diagnosing a scabies mite infection of the present invention may further include determining that the scabies mite is infected when the cox1 gene is detected. The information providing method provides information on whether the cox1 gene derived from the scabies mite is detected from a biological sample isolated from a target subject, and when the cox1 gene is detected in the sample, the subject from which the sample is isolated is infected with scabies mite or It can be diagnosed as scabies.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1: Microscopic examination using dermoscopy

Dermoscopy examination and microscopy were previously described in Annals of dermatology. 2012;24(2):194-199.]. To confirm the triangular structure corresponding to the anterior part of the tick (a trace similar to that of a jet airliner), a dermoscope (Dermlite III; 3GenInc., San Juan Capistrano, CA, USA) was used, between the fingers and between the fingers at 10x magnification of the dermoscope. The body parts of patients with corrugated areas such as sides, wrists, armpits, navel, buttocks, and groin were investigated.

Scratching of the triangular structure was examined for the presence of mites or eggs under a 100 magnification microscope by a dermatologist with more than 10 years of experience in skin and microscopic identification of scabies. If at least one tick or egg was found under a microscope, the patient was clearly diagnosed as infected with the scabies mite, which was designated as a “microscopic positive” patient. A "microscopic examination negative" patient is a case where symptoms of scabies are suspected, but the skin abrasion specimen is negative. The skin slapping specimens on glass slides observed under the microscope were transferred to a sterile 1.5 mL microcentrifuge tube and stored at -80°C for PCR.

On the other hand, microscopically positive patients were treated with permethrin cream (Daewoong Pharmaceutical, Seoul) on the neck two days a week in a row, and then received a dermoscopy test every week. If the dermoscopy test was positive, the treatment was repeated, and if it was negative, the treatment was stopped. If the dermoscopy test was negative for at least 2 weeks, it was judged that the scabies had been cured. In addition, scraping samples for PCR were collected from all patients at each follow-up visit.

Example 2: DNA sample extraction

DNA extraction was performed using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Specifically, the sample was digested with 180 μL of buffer ATL buffer (Qiagen) and 20 ul of proteinase K (Qiagen) at 56° C. for 1 hour, 200 μl of buffer AL (Qiagen) was added and 70° C. for 10 minutes. was incubated in After purification, total DNA was eluted in 60 μl of buffer AE (Qiagen).

Example 3: Primer and Probe Design

Primer sequences used in Examples and Experimental Examples of the present specification are shown in Table 1 below.

PCR type and primer name double PCR
reaction step PCR program Sequence(5'--> 3') order
number Conventional Nest-scab-F2 Primary 95° C., 10 minutes (once);
95°C, 1 minute->53.3°C, 1 minute->72°C, 1 minute (repeat 40 times); 72°C, 10 minutes TTT TTC GGA CAC CCA GAA GT One Nest-scab-R1 Primary CCA ATT GCC CAA TAT ATA GAA GGA 2 q-scab-F Secondary 95° C., 10 minutes (once);
95°C, 1 minute->62°C, 1 minute->72°C, 1 minute (repeat 40 times); 72°C, 10 minutes GCT ATG ATT TCT ATT GCA ACC T 3 q-scab-R Secondary ACT CCT GTA GGA ACA GCA ATA 4 Real-time Nest-scab-F2 Primary 95° C., 10 minutes (once);
95°C, 1 minute->53.3°C, 1 minute->72°C, 1 minute (repeat 40 times); 72°C, 10 minutes TTT TTC GGA CAC CCA GAA GT One Nest-scab-R1 Primary CCA ATT GCC CAA TAT ATA GAA GGA 2 q-scab-F Secondary 95° C., 10 minutes (once);
95℃,1min->58.3℃,1min->72℃,1min (repeat 40 times) GCT ATG ATT TCT ATT GCA ACC T 3 q-scab-R Secondary ACT CCT GTA GGA ACA GCA ATA 4 probe Secondary /56-FAM ^a /AC TGT AGG T/ZEN/T TAG ATG TTG ATA CCC GAG C/3IABkFQ ^b 5

^a 6-FAM, 6-carboxyfluorescein fluorescent dye ^b 3IABkFQ, Iowa black quencher

External primers (Nest-scab-F2 and nest-scab-R1) were [ The British journal of dermatology. 2018;179(4):889-895], which amplifies the 320-base pair (bp) fragment of the human scabies mite (Sarcoptes scabiei var. hominis) cox1 gene. The internal primers (q-scab-F and q-scab-R) that amplify the 128-bp DNA fragment of the cox1 gene were newly identified using the primer3plus web-based program (www.bioinformatics.nl/primer3plus) [ The British journal of dermatology. 2018;179(4):889-895] (GenBank Accession No. MF984353.1). A Taqman probe that identifies a 29-bp fragment of the cox1 gene was prepared using the primerquest tool (www.idtdna.com/pages/tools/primerquest). Dermatophagoides pteronyssinus, Dermatophagoides farinae, Demodex folliculorum, Demodex brevis, microspores disclosed in the NCBI sequence database with the prepared primer and probe sequences (Microsporum canis) and Trichophyton rubrum (Trichophyton rubrum) between the primer and probe sequences and the sequence of the cox1 gene was not found homology.

Example 4: Double PCR amplification and sequencing

The double polymerase chain reaction method (nested PCR) was carried out by performing the first PCR reaction and the second PCR reaction, that is, two PCR reactions.

Specifically, first, to perform the first PCR reaction, 2 μl of DNA extract, 1 μl of each 10 pM primary forward and reverse primers (primary primer pair), 6 μl of nuclease-free water (Integrated DNA Technologies, Coralville, IA, USA) and 10 μl of a PCR master mix (H-star PCR Master Mix, BIOFACT) were prepared and reacted with 20 μl of a primary PCR reaction mixture. Meanwhile, thermal cycling was performed using an automatic thermal cycler (LifePro, Bioer, Hangzhou, China).

Next, to perform a secondary PCR reaction, 1 μl of the primary PCR product, 1 μl each of 10 pM secondary forward and reverse primers (secondary primer pair), 7 μl of water, and 10 μl of PCR master mix 20 μl of a secondary PCR reaction mixture containing Meanwhile, the PCR program for each primer pair is shown in Table 1 above.

In order to confirm the amplification result by the PCR reaction, the following experiment was performed. Specifically, 4 μl of the amplified product was run in parallel on a 1.7% (w/v) agarose gel with a 100-bp DNA ladder (BIOFACT) and electrophoresed in 1 X Tris. In addition, the gel was stained with a fluorescent dye (EcoDye Nucleic Acid Staining Solution, BIOFACT) in acetate-EDTA buffer at 120 V for 40 minutes, and pictures were taken under UV irradiation.

Positive and negative controls were parallelized in each run. The positive control group was skin abrasions from patients with microscopic scabies, and the negative control group was patients with other skin diseases such as allergic contact dermatitis, atopic dermatitis, seborrheic dermatitis, thermal sclerosis, psoriasis, fungal layer, iris color and all dermatosis. skin abrasions of PCR reagents, water, DNA extracted from house dust mites, and DNA extracted from samples from patients infected with pure skin pathogens (M. canis, T. rubrum) and hair mite (D. folliculorum) were also used to test the specificity of double PCR.

At this time, both strands of the PCR products were sequenced by an ABI PRISM 3730xl DNA analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions using internal PCR primers specific for each PCR product. analyzed. DNA sequences were analyzed to confirm their identity by BLASTn search against NCBI's online non-redundant nucleotide collection (nr/nt) database.

Example 5: Dual real-time qPCR

DNA extracts from scraping specimens identified by the presence of mites under the microscope were amplified by double PCR. The final product was inserted into a plasmid vector and cloned using the All in One PCR cloning kit (BIOFACT). After propagation and purification of the plasmid, the DNA copy number per reaction was calculated from the A260 optical density measurement of the plasmid DNA concentration. Each qPCR run involved 6 serial 10-fold dilutions of plasmid DNA ranging from 7.22 X 10 9 to 104 copies of DNA extracted per reaction. The amount of scabies mites ( Sarcoptes scabiei) in each specimen was estimated by comparing the cycle threshold (CT) number with the number prepared from serial dilutions of plasmid DNA. The detection limit was 10 copies per reaction mixture.

For dual real-time qPCR, 1 μl of primary PCR product, 1 μl each of 10 pM new forward and reverse internal primers (BIONEER), 0.5 μl of 10 pM probe (integrated DNA technology), 6.5 μl of water and 10 μl of PCR master Mix (Multi-star real-time PCR master mix, BIOFACT) was mixed to prepare a reaction mixture. Positive and negative controls were also included in each run. Thermal cycling was performed in an automated CFX-96 real-time PCR system (Biorad, Hercules, CA, USA), and the program for real-time qPCR is shown in Table 1.

Example 6: Statistical Analysis

To analyze the clinical characteristics of patients, Student's t-test, chi-square test, Kruskal-Wallis test and linearity by linear connection were used. McNemar's test was used to compare the results of the double real-time PCR and DS tests in the first diagnostic test. A 95% confidence interval (CI) for differences between JEN groups was calculated using Student's t-test. The non-inferiority margin is estimated at 10 percentage points. The log-modified tick DNA load was then compared by Wilcoxon-signed rank test. P-values less than 0.05 were considered statistically significant and statistical analysis was performed using PSS software (version 24.0, IBM, Armonk, NY, U.S.A.).

Experimental example

A total of 150 patients with suspected scabies infection were enrolled between July 2018 and October 2019. The average age of these patients was 51.2 years (7 months to 97 years) and the male to female ratio was 1:1. On microscopic examination, 67 patients were diagnosed with definite scabies (DS-positive), and 83 patients were DS-negative. 42 out of 67 cases (FU group) were identified as scabies were followed every 7 days until microscopy turned negative for at least 2 weeks. Of the 67 patients, 25 (in the FU_loss group) were poorly followed. Demographics were not significantly different in the three groups, FU, FU_loss, and DS-negative group (P > 0.05; Table 2 - Demographics of participants according to microscopy results)

demographics DS ^a positive (n = 67) DS negative (n = 83) P value Age (years), mean ± SD (range) 53.23±22.44 49.47±21.29 0.295 last name, n male 34 37 woman 33 46 Male: Female Ratio 1: 0.97 1:1.24 0.452 FU ^b (n = 42) FU_loss(n = 25) age 53.66±24.09 50.52±19.82 0.327 ^c last name, n male 20 14 woman 22 11 Male to female ratio 1:1.1 1: 0.79 0.613 ^c

* a: Skin abrasion and microscopic examination b: Follow-up

Differences between c:FU, FU_loss and DS negative groups

Meanwhile, all 67 microscopically positive patients were positive for PCR. Of the 83 samples (14.1%) of patients suspected of scabies but diagnosed as microscopically negative, 11 (14.1%) were positive using cox1 overlap real-time PCR. These 11 patients were treated with 5% permethrin cream and successfully improved, showing that they had scabies. In addition, 4 of 83 patients responded to cox1 overlap real-time PCR but were considered negative because the S. scabiei copy number was lower than 10 copies of the cut-off value and improved after anti-inflammatory treatment. All samples positive for cox1 nested real-time PCR were also positive for cox1 nested conventional PCR with identical primers. Sequence analysis of these PCR products (GenBank accession numbers MT133585 to MT133651) showed 98-100% nucleotide sequence identity to the human scabiei var. cox1 gene (GenBank accession numbers AY493388.1 and MF984353.1).

The sensitivity of DS is 85.90% (95% CI 76.17 - 92.74) when the sensitivity of cox1 nested real-time PCR is considered 100% (95% CI 95.38-100). The difference between them was statistically significant (P = 0.001) (Table 3, results of microscopy and Cox1 overlap real-time PCR in scabies diagnosis).

Cox1 double real-time PCR results Number of samples with microscopic results Microscope sensitivity
Sensitivity of the microscopy ^a P-value
P-value ^b positivity voice Total positivity 67 0 67 85.90% 0.001 voice 11 72 83 Total 78 72 150

* a: Sensitivity was calculated assuming that cox1 nested real-time PCR has a sensitivity of 100%. b: The difference between the two methods is statistically significant (P <0.05)

When the non-inferiority margin was considered to be 10% (-Δ = -0.1), the 95% CI of the difference between microscopy and double real-time PCR results was lower than -0.1 (-0.942 to -0.793). All negative controls: 1 patient with atopic dermatitis, 1 patient with allergic contact dermatitis, 1 patient with seborrheic dermatitis, 1 patient with eczema dermatitis, 1 patient with psoriasis, 1 patient with sarcoma, 1 patient with erythema, 1 patient with erythema Total DNA was extracted from samples of In addition to pure cultures of M. canis, T. rubrum, D. farinae and D. pteronyssinus, demodecidosis is included. All these samples are negative for nested real numbers.

42 patients were followed up as appropriate every 7 days until consecutive microscopy at least twice a week was negative. The mean number of follow-up visits excluding the date of diagnosis was 3.40 (range 2 to 6). The mean treatment week for these patients was 2.02 (range 1-4). The mean number of log10-modified DNA copies of S. scabiei at diagnosis was 9.22 (95% CI 8.82 - 9.62). between days 0 and 7 (mean 5.62 (95% CI 4.33 - 6.91)), between days 7 and 14 (mean 3.72 (95% CI 2.60 - 4.83), between days 14 and 21 (mean 1.76 (95% CI)) 1.02 -2.51) there was a statistically significant decrease, but the difference was not significant between days 21 and 28 (mean 1.76 (95% CI 0.66 - 2.82)) (P = 1.000) (Table 4 - Parasitic load changes during follow-up period) , Figure 1 - Parasitic load changes during follow-up after initiation of treatment).

Log10 copies per reaction (95% CI) P-value Day 0 (n = 42) 9.22 (8.82 - 9.62) Day 7 (n = 42) 5.62 (4.33 - 6.91) 0.000 ^a Day 14 (n = 42) 3.72 (2.60 - 4.83) 0.013 ^b Day 21 (n = 37) 1.76 (1.02 - 2.51) 0.005 ^c Day 28 (n = 18) 1.76 (0.66 - 2.82) 1.000 ^d

* a: the difference between days 0 and 7 was statistically significant (P <0.05)b: the difference between days 7 and 14 was statistically significant (P < 0.05)

c: The difference between days 14 and 21 was statistically significant (P < 0.05).

d: The difference between days 21 and 28 was not statistically significant. (P > 0.05)

On the other hand, Figure 3 shows the qPCR test during the follow-up period, and in 34 of 42 patients, no tick DNA was detected during the follow-up period after treatment, but tick DNA was detected up to 1 week for 2 weeks (4.76%), 2 weeks 4 cases (9.52%) by the time of diagnosis and 2 cases (4.76%) by 3 weeks were detected. The mean tick DNA load during this period was 4.2% (95% CI 0.93 - 7.47) relative to the peak. None of these patients relapsed clinically and microscopically.

Figure 2 shows the sequential changes of parasitic hatching and DS test results and permethrin treatment in a 71-year-old male patient. He was treated with oral and topical corticosteroids for several weeks at a private dermatology clinic for systemic itching, but no improvement. The DS test on day 0 showed several mites and eggs. He was instructed to use a 5% permethrin cream on his neck for 2 consecutive days for a week, and his symptoms improved. DS-test was negative on day 7 and remained until day 77. However, on day 91, the DS test turned positive and he was treated again. On days 98 and 105, the DS test remained positive and treatment was repeated. On day 112, the DS test turned negative and remained on days 119 and 126. He was followed up until 8 months later, but there was no recurrence. Changes in the tick DNA load were similar to the DS-test results. DS test results at 91 days were considered relapse rather than treatment failure because there was an 11-week interval from the first treatment (14 days) to relapse (91 days).

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION, HALLYM UNIVERSITY <120> Method of providing information for diagnosis and treat monitoring of Sarcoptes scabiei infection <130> P201070 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nest-scab-F2 <400> 1 tttttcggac acccagaagt 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Nest-scab-R1 <400> 2 ccaattgccc aatatata agga 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> q-scab-F <400> 3 gctatgattt ctattgcaac ct 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> q-scab-R <400> 4 actcctgtag gaacagcaat a 21 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 5 actgtaggtn tagatgttg atacccgagc 30

Claims (12)

extracting a DNA sample from the biological sample isolated from the subject;
amplifying by a double PCR reaction; and
Including; performing double real-time qPCR;
When the cox1 gene is detected by amplification by the double PCR reaction, it is determined that the scabies mite is infected,
The double real-time qPCR is to extract DNA from a biological sample of an individual determined to be infected with the scabies mite, insert it into a plasmid vector, proliferate and purify it, and then measure the DNA concentration of the plasmid to monitor whether or not to treat the scabies mite,
The double real-time qPCR is a primary primer pair comprising the sequence of SEQ ID NOs: 1 to 2; a secondary primer pair comprising the sequence of SEQ ID NOs: 3 to 4; and a probe comprising the sequence of SEQ ID NO: 5; a method for providing information for diagnosis and treatment monitoring of scabies mite infection. delete According to claim 1,
The cox1 gene is derived from human scabies mite (Sarcoptes scabiei var hominis), a method for providing information for diagnosis and treatment monitoring of scabies mite infection. delete delete According to claim 1,
The first pair of primers is for detecting the cox1 gene derived from Sarcoptes scabiei. 7. The method of claim 6,
The cox1 gene is derived from human scabies mite (Sarcoptes scabiei var hominis), a method for providing information for diagnosis and treatment monitoring of scabies mite infection. A primary primer pair comprising the sequence of SEQ ID NOs: 1 to 2 and for detecting the cox1 gene;
a secondary primer pair comprising the sequence of SEQ ID NOs: 3 to 4; and
A diagnostic kit, including a double real-time qPCR kit comprising a probe comprising the sequence of SEQ ID NO: 5, and for monitoring whether or not the scabies mite is treated. delete delete delete delete

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