KR101081602B1 - Primer sets for detection of Cryptosporidium and a composition comprising the same - Google Patents

Abstract

The present invention relates to a Cryptosporidium diagnostic primer, a diagnostic method using the primer, and a diagnostic composition comprising the primer.

Cryptosporidium parvum, nested PCR, COWP gene

Description

Cryptosporidium diagnostic primer and its composition {Primer sets for detection of Cryptosporidium and a composition comprising the same}

The present invention relates to a Cryptosporidium diagnostic primer, a diagnostic method using the primer, and a diagnostic composition comprising the primer.

Cryptosporidium parvum ( Apicomplexa: Cryptosporididae) is a protozoan parasite mainly found in the gastrointestinal tract of vertebrates. C. parvum is transmitted to 79 mammals, including humans, causing acute gastroenteritis. Unlike most parasites, C. parvum has low host specificity in mammals and can therefore be cross-infected with multiple host families.

Cryptosporidiosium is transmitted to the follicular sac via the feces-oral route. Contaminated water, intimate human-human contact, and contact with infected animal secretions, zoo animals or poultry can cause infection of the parasite. In exogenous periods outside the host, Cryptosporidium exists as sporulated follicular cysts. The follicle consists of four sporozoite inside two rigid walls. The follicular wall is sealed at the inner and outer layers and at one end. During the excystation, the suture melts, providing an entrance for the species to exit the follicular sac. When ingested by a suitable host, living organisms, which are bark larvae, live in the gastrointestinal or respiratory tract cells. After reproduction, resporulation occurs into the follicle. In the gastrointestinal tract, follicles are secreted together with feces, and the respiratory tract is released into the body as respiratory and nasal secretions.

Infection by Cryptosporidium usually occurs in all countries, both developed and underdeveloped. The slightly higher incidence of Cryptosporiidiosis in underdeveloped countries is due to poor hygiene, malnourished contaminated drinking water and close contact with infected people and animals.

The most common clinical sign associated with Cryptosporidiosis is diarrhea and it can be severe, causing weight loss and dehydration. Other common clinical symptoms include abdominal pain, high fever, nausea, vomiting, headache, fatigue, muscle pain and loss of appetite. Infection with Cryptosporidium is usually in the small intestine, but often occurs in the lungs, esophagus, stomach and other organs. Clinical symptoms associated with the parasitic infection depend on the infected organ.

The range of symptoms and severity of the disease varies greatly from person to person and can be life threatening. Symptoms of acute enteritis often last 1 to 2 weeks in immunologically healthy individuals. Cryptosporidium presents a major threat to AIDS patients, malnutrition patients, patients with innate immune deficiency, and those taking immunosuppressants.

All infections of Cryptosporidium are initiated by the ingestion or inhalation of the follicle. Because the parasites are infected in the form of follicular cysts, the follicles survive in severe cold conditions and are often resistant to natural disorders and chemical fungicides. The presence of exogenous follicular spores also makes the protozoan parasite more resistant to conventional water treatment. Methods for preventing or limiting the spread of infection focus on removing or reducing infectious follicles in the environment. In humans, sterilization processes focus on minimizing the transmission of humans to humans and efficiently dealing with contamination of the water supply.

The recent outbreak of the development of large waterborne diseases notes the importance of understanding their transmission through the environment. Surface water can be naturally contaminated mainly by infected animal secretions. Many waste treatment processes can create polluted rivers or rivers. Excretion contamination in waterways causes large numbers of C. parvum infections. And water contaminated by these practices can cause contamination of food grains during contamination or tillage of drinking water.

Diagnosis of C. parvum infection generally relies on microscopic detection of follicular cysts, but it does not provide information on the infected species and is a difficult problem even for highly experienced experimental researchers (Coupe S, Sarfati C, Hamane). S, Derouin F. J Clin Microbiol 2005; 43: 1017-1023.).

Another method for detecting follicular follicle follicles is described in US Pat. No. 6,146,838 to Williams et al. And US Pat. No. 6,475,747 to Tsang et al., Both patents derived from It is described to dissolve antigens. US Pat. No. 6,146,838 describes in particular dissolution in the presence of bile salts and / or ionic detergents. U.S. Patent No. 6,475,747 describes follicular cyst lysis using zwitterionic detergents. Both patents also describe the detection of lysed antigens using electrochemical luminescence (ECL) immunoassays.

Therefore, a rapid, simple, specific and sensitive method is needed for the detection of C. parvum . Molecular detection techniques, such as PCR-based methods, offer many advantages over microscopic methods .

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and the object of the present invention is to provide a method for detecting Cryptosporidium follicles (oocysts) that is rapid, specific and highly sensitive.

Still another object of the present invention is to provide a primer used for detection of the Cryptosporidium follicles (oocysts).

In order to achieve the above object, the present invention

Selective detection by nested polymerase chain reaction (nested PCR) using oligonucleotides consisting of the nucleotide sequences of SEQ ID NO: 3 to SEQ ID NO: 4 as primers for the follicular wall protein (COWP) gene of Cryptosporidium Provided are a method for detecting Cryptosporidium using a sted polymerase chain reaction.

In one embodiment of the present invention, the PCR used in the detection method of the present invention further comprises a nucleotide of SEQ ID NO: 1 to SEQ ID NO: 2.

In a preferred embodiment of the present invention, the Cryptosporidium is preferably Cryptosporidium parvum, but is not limited thereto.

In another aspect, the present invention provides a primer for Cryptosporidium detection, characterized in that the oligonucleotide represented by SEQ ID NO: 3 to SEQ ID NO: 4.

In one embodiment of the present invention, the primer of the present invention further comprises a nucleotide of SEQ ID NO: 1 to SEQ ID NO: 2.

The present invention also provides a composition for detecting Cryptosporidium using an oligonucleotide primer represented by SEQ ID NO: 3 to SEQ ID NO: 4 as an active ingredient.

In one embodiment of the present invention, the composition of the present invention further comprises a nucleotide primer of SEQ ID NO: 1 to SEQ ID NO: 2.

In another aspect, the present invention provides a nested PCR kit for detecting Cryptosporidium using an oligonucleotide primer represented by SEQ ID NO: 3 to SEQ ID NO: 4 as an active ingredient.

In one embodiment of the present invention, the nested PCR kit primer of the present invention further comprises a nucleotide primer of SEQ ID NO: 1 to SEQ ID NO: 2.

Hereinafter, the present invention will be described.

Improved methods for the detection of Cryptosporidium follicles (oocysts) in environmental and clinical samples are urgently needed to improve the detection of Cryptosporidiumosis.

We believe in Cryptosporidium parvum. The sensitivity of 7 PCR primer sets for detection was compared. Each target gene was amplified by PCR or nested PCR with a series of diluted DNA extracted from purified C. parvum follicular follicles. The target genes Cryptosporidium I cyst wall proteins include;; (SSU rRNA small subunit ribosomal RNA) and other rRNA (Cryptosporidium oocyst wall protein COWP), small subunit ribosomal RNA. The limit of detection by the PCR method was in the range of 10 ³ to 10 ⁴ follicles, and the nested PCR method could detect 10 ⁰ to 10 ² follicles.

Nested PCR primer sets specific for the COWP gene showed the highest sensitivity among the other primer sets tested in the present invention and thus may be useful for the detection of C. parvum .

In the present invention, the sensitivity of several PCR target gene primer sets for C. parvum detection was compared.

PCR performed in two steps in the present invention is a so-called "double PCR (Nested PCR)", which is briefly described as follows.

First, a primer set (first primer set, outer primer) that forms amplification product (first amplification product) larger than the final target DNA by binding complementarily with a specific DNA sequence located outside the target DNA sequence. ) Performs the first nucleic acid amplification. A primer set for forming a second amplification product which is the final target DNA by binding complementarily with a specific sequence located inside the first amplification product for the first amplification product generated using the first primer set. A second nucleic acid amplification is performed using (second primer set, inner primer). In this case, the primers (5 'primer and 3' primer) constituting the second primer set specifically bind to both ends of or near the ends of the final target DNA, respectively.

That is, the double PCR used in the present invention performs a first amplification using a primer set that complementarily binds to a specific sequence existing outside the final target DNA to form a first amplification product, and Continuously performing a second amplification using a primer set that complements a particular sequence present therein, wherein the first amplification product by the first primer set comprises a second amplification product by the second primer set do. In the case of using such a double PCR, since the second amplification process is continuously performed on the first amplification product formed by the first amplification process, it is necessary to repeat the PCR cycle indefinitely even when a small amount of nucleic acid is included in the sample. There is no. Thus, since it is possible to prevent amplification of nucleic acid sequences other than the target nucleic acid which may be caused by an increase in the PCR cycle, it is possible to greatly improve the sensitivity and specificity of the finally formed amplification products.

In the present invention, the first primer set and the outer primer use Cry-15 and Cry-9 primers as set out in SEQ ID NOs: 1 and 2, and Cowpnest-F1 and cowpnest-R2 as set out in SEQ ID NOs: 3 and 4 as second primer sets. It was.

In this regard, in accordance with a preferred embodiment of the present invention, the complementary binding to the follicular wall protein (COWP) gene region of Cryptosporidium parvum ( Cryptosporidium parvum) of SEQ ID NO: 1 (or sequence complementary to SEQ ID NO: 1) Oligonucleotides of an oligonucleotide and SEQ ID NO: 2 (or a sequence complementary to SEQ ID NO: 2)

After forming a first amplification product using a set of primers consisting of an oride, it is complementarily bound to a specific sequence located inside the first amplification product, SEQ ID NO: 3 (or complementary to SEQ ID NO: 3) An oligonucleotide of SEQ ID NO: 4 and SEQ ID NO: 4 (or

By using a second set of primers consisting of oligonucleotides of the sequence complementary to SEQ ID NO: 4, the sensitivity was improved by about 1,000 to 10,000 fold over the conventional method for the detection of small spores.

In the present invention, we describe C. parvum . A set of highly sensitive primers specific for the COWP gene that can be used for detection has been developed. This highly sensitive nested PCR method will be useful to enable detection of Cryptosporidium in patients with a low number of follicular cysts and may be a valuable method for detection in environmental and clinical samples.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, the scope of the present invention is not limited by the following examples.

Example 1: C. parvum  Isolation and Extraction of DNA

Follicle sacs of C. parvum (KKU isolates) were immunized with dexamethasone phosphate disodium salt (Sigma, St. Louis, MO, USA) randomly given to drinking water at a dose of 10 mg / ml and the pathogen-free C57BL female mice (Yang S, Healey MC. J Parasitol 1993; 79: 626-630). Follicle cyst purification was performed according to the method of Petry et al. (Petry F, Robinson HA, McDonald V. J Clin Microbiol 1995; 33: 1922-1924). Purified follicles were surface-sterilized by placing in 10% sodium hypochlorite solution for 10 minutes and maintained at 4 ° C. in filtered (0.22 μm) distilled water for 2 weeks.

For the extraction of C. parvum DNA, 10 ⁷ The isolated follicles were resuspended in the ASL Lysis buffer included in the QIAquick stool mini kit (QIAGEN Inc, Valencia, CA, USA). The samples were incubated at 70 ° C. for 30 minutes. The procedure was performed according to the manufacturer's recommendation. The extracted DNA was used as a template for PCR.

Example 2: Amplification by PCR and Nested PCR of DNA extracted from Cryptosporidium parvum follicle

All primer sets specific to the C. parvum target gene used in the present invention were prepared in Bioneer, Daejeon, Korea, It shows in Table 1 (Table 1). The position of the primer sequence in each specific C. parvum gene is shown in FIG. Primer pairs, cowpnest-F1 and cowpnest-R2, were designed for nested PCR specific to the COWP gene. PCR was performed with four primer sets including Cry-15, Cry-19 (COWP), BcowpF, BcowpR (COWP-1), rRNAF, rRNAR (SSU rRNA), SB012F, SB012R (rRNA). PCR amplification was performed with template DNA (5 μl of genomic C. parvum DNA at the same concentration as the number of C. parvum follicles diluted from 10 ⁵ follicles), 10 mM-HCl (pH 8.3), 2.5 mM MgCl ₂ , 200 μM each dATP, dCTP, dGTP, and dTTP, 25 pmole each primer and 2.5 μL Taq DNA polymerase (Promega, Madison, WI, USA), respectively, were performed in 50 μl volumes. All amplifications were performed in a Perkin-Elmer DNA thermal cycler (Model: 2400, Wellesley, Mass., USA) with initial denaturation conditions at 94 ° C for 5 minutes, denaturation at 30 cycles of 94 ° C for 50 seconds, at 30 specified temperatures. Second annealing (Table 1), and 50 seconds of expansion at 72 ° C., final expansion was performed at 72 ° C. for 10 minutes. For Nested PCR, 2 μl of the original purified PCR product was used as template. The nested PCR cycling conditions were the same as those used for PCR amplification except that the annealing of the nested primers was performed at each temperature listed in Table 1. Amplified DNA fragments were electrophoresed on 1.5% (w / v) agarose gel, stained with ethidium bromide (0.5 μg / ml) and visualized under UV light system (Vilber Lourmat, France).

From the PCR amplification, we obtained products of the following predicted sizes: 550 bp (COWP gene), 769 bp (COWP gene-1), 1,325 bp (SSU rRNA gene), and 433 bp (rRNA gene) (FIG. 2). COWP gene-1 is a larger PCR product than the COWP fragment amplified by primers Cry-15 and Cry-19 and it contains this site. We also obtained the following predicted PCR products by Nested PCR amplification: 311 bp (COWP gene), 550 bp (COWP gene-1), and 826 bp (SSU rRNA gene) (FIG. 2).

The lowest limit of detection of the PCR amplification is been 10 ³ to 10 ⁵ I cysts in all the primer set. However, greater levels of sensitivity were observed in nested PCR amplification and their detection limits were as low as single follicular cysts (FIG. 2, Table 2). The most sensitive nested PCR target gene was COWP, and the primers cowpnest-F1 and cowpnest-R2 were able to detect single follicular cysts. The detection limit for the rRNA gene was 10,000 follicles when SB012F and SB012R primers were used for PCR amplification (FIG. 2D). Nested PCR was not performed on that primer set.

Although stool microscopy was useful for the identification of infections, this method was relatively insensitive when small amounts of follicles were secreted or the period of follicle divergence was short. Thus many infections can deviate from microscopic detection (McCluskey BJ, Greiner EC, Donovan GA. Vet Parasitol 1995; 60: 185-190).

In the present invention, the nested PCR reaction specific to the COWP gene had an obvious effect compared to other primers. In particular, the combination of cowpnest F1 and cowpnest R2 primers had great sensitivity and could detect as few as one follicle. Another primer pair for the COWP gene, BcowpF and BcowpR, was designed to produce larger fragments of this gene containing sites amplified by cowpnest-F1 and cowpnest-R2. The sensitivity of nested PCR using this primer set was 100 times lower than the smaller product of the COWP gene obtained by cowpnest-F1 and cowpnest-R2.

The number of follicular cysts used to generate template DNA in the present invention was indirectly calculated using diluted DNA extracted from purified follicular cysts.

Target genes primer order Expected product size
(bp) Annealing temperature
(℃) COWP * Cry-15
Cry-9 5'- GTA GAT AAT GGA AGA GAT TGT G-3 '(SEQ ID NO: 1)
5 '-GGA CTG AAA TAC AGG CAT TAT CTT G-3' (SEQ ID NO: 2) 550 52 COWP cowpnest-F1
cowpnest-R2 5 '-TGT GTT CAA TCA GAC ACA GC-3' (SEQ ID NO: 3)
5'- TCT GTA TAT CCT GGT GGG C-3 '(SEQ ID NO: 4) 311 60 COWP BcowpF
BcowpR 5 '-ACC GCT TCT CAA CAA CCA TCT TGT CCT C -3' (SEQ ID NO: 5)
5 '-CGC ACC TGT TCC CAC TCA ATG TAA ACC C -3' (SEQ ID NO: 6) 769 55 SSU rRNA rRNA F
rRNA R 5'- TTC TAG AGC TAA TAC ATG CG-3 '(SEQ ID NO: 7)
5'- CCC TAA TCC TTC GAA ACA GGA-3 '(SEQ ID NO: 8) 1325 55 SSU rRNA nest rRNA F
nest rRNA R 5'- GGA AGG GTT GTA TTT ATT AGA TAA AG-3 '(SEQ ID NO: 9)
5'- AAG GAG TAA GGA ACA ACC TCC A-3 '(SEQ ID NO: 10) 826 55 rRNA SB012F
SB012R 5'- CTC CGT TCG ATG ATG CAG ATG-3 '(SEQ ID NO: 11)
5'- CGG CCC CTG TAG AAA TAA GTC A-3 '(SEQ ID NO: 12) 433 51

Table 1 shows primer sets for PCR and Nested PCR specific for C. parvum genes. * COWP in Table 1 represents Cryptosporidium oocyst wall protein.

C. parvum
Number of follicles COWP
PCR COWP
nested
PCR BCOWP *
PCR BCOWP **
nested PCR SSU rRNA
PCR SSU rRNA
nested
PCR SB012
PCR 10 ⁵ ++ ^b +++ ^a ++ +++ ++ +++ ++ 10 ⁴ + ^c +++ ++ +++ + +++ + 10 ^{3 -d} +++ + +++ - +++ - 10 ² - +++ - +++ - +++ - 10 ¹ - +++ - - - +++ - 10 ⁰ - +++ - - - - -

Table 2 shows the difference in sensitivity between the various primer sets for PCR and Nested PCR of C. parvum .

In Table 2, BCOWP PCR * indicates PCR performed with BcowpF and BcowpR primers, BCOWP nested PCR ^** indicates Nested PCR performed with Cry-15 and Cry-19 primers after BCOWP PCR, and ^a +++ ethidium bromide stained—the strong sensitivity of the DNA band determined by agarose gel electrophoresis, ^b ++ is the intermediate sensitivity of the DNA band, ^c + is the weak sensitivity of the DNA band, and ^d — ethidium bromide stained. By agarose gel electrophoresis means that the DNA band is not visible to the naked eye.

1 shows the location of primer pairs specific for Cryptosporidium parvum gene for PCR and nested PCR. Cryptosporidium parvum target genes for PCR were obtained from each GenBank accession no. (COWP, Z22537 SSU rRNA, AF093489 SB012, AF161076).

2 is amplification by PCR and nested PCR of Cryptosporidium parvum DNA extracted from purified Cryptosporidium parvum follicle. The top gel shows amplification by PCR, and the bottom gel shows amplification by nested PCR using a PCR product amplified with template DNA. M: 100 bp DNA ladder marker (Promega). A: COWP gene, B: BCOWP gene, C: SSU rRNA gene, D: SB012 rRNA gene.

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Claims (10)

Primer for the follicular wall protein (COWP) gene of Cryptosporidium (Cryptosporidium) consisting of an inner primer pair having a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 and an outer primer pair having a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 Cryptosporidium detection method using nested polymerase chain reaction selectively detects by nested polymerase chain reaction using inner and outer primer pairs delete The method of claim 1, wherein the Cryptosporidium is Cryptosporidium parvum. SEQ ID NO: 3 and SEQ ID NO: 4; or A primer pair for detecting Cryptosporidium having a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2. delete The primer pair of claim 4, wherein the Cryptosporidium is Cryptosporidium parvum. Cryptosporidium detection composition comprising the primer pair of claim 4 as an active ingredient. The composition of claim 7, wherein the Cryptosporidium is Cryptosporidium parvum. Cryptosporidium detection nested polymerase chain reaction (PCR) kit using the primer pair of claim 4 as an active ingredient. 10. The method according to claim 9, wherein the Cryptosporidium (Cryptosporidium) is a nested polymerase chain reaction (PCR) kit for detecting Cryptosporidium, characterized in that Cryptosporidium parvum.

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