TWI681057B - Primer pair for detecting ureaplasma urealyticum serovar 10 and kit thereof - Google Patents

Abstract

The present invention provides a primer pair for detecting Ureaplasma urealyticum serovar 10 (UU-10), consisting of an oligonucleotide of SEQ ID NO: 1 and an oligonucleotide of SEQ ID NO: 2. The primer pair of the present invention may be used in the fast and accurate examination of UU-10 infection in the urogenital system, as well as in evaluating the risk of complications resulted from UU-10 infection. Also provided is a kit for detecting UU-10 by PCR, comprising a primer pair of the present invention.

Description

Primer pair and set for detecting ureaplasma serotype 10

The invention relates to a nucleic acid detection method for ureaplasma urealyticum serotype 10, in particular to a primer pair and its kit for detecting ureaplasma urealyticum serotype 10.

Uroplasma infection is the most common bacterial infection of the urogenital system. After infection, it often causes diseases related to the urogenital system such as prostatitis, vaginitis, urethritis, and infertility. If a pregnant woman is infected with uroplasma, spontaneous abortion, premature birth, Chorioamnionitis and other diseases will increase the risk [1], if further enter the baby's mouth, nose, and lungs through amniotic fluid, it may cause bronchopulmonary dysplasia (BPD), respiratory distress syndrome (respiratory distress syndrome) syndrome, RDS) or even neonatal death [1-3].

Because of its long incubation period and its asymptomatic status, it is estimated that the proportion of urinary tract infections in healthy adult females is about 40 to 80% [2, 4]. Pregnant women, due to hormonal changes during pregnancy, suppress the immune response, leading to more It is susceptible to urinary bacterium infections, and the proportion of vertical infections from mothers to newborns reaches 90% [2].

Ureaplasma spp. is classified as Mollicutes, Mycoplasmatales, Mycoplasmatales, Mycoplasmataceae, Genus ureaplasma , including Ureaplasma parvum and Ureaplasma spp. Ureaplasma urealyticum [2], currently can be divided into 14 serotypes according to the multiple banded antigen (MBA) on the surface of urea bacteria [2, 5], 4 of which belong to microurea Serotypes (serotypes 1, 3, 6 and 14), and the other 10 species belong to ureaplasma urealyticum (serotypes 2, 4, 5 and 7-13) [1].

Uroplasma bacteria of specific serotypes are often discussed for their prevalence, drug resistance, pathogenicity, invasiveness, preterm birth complications and the association of specific diseases, etc. [6-8], Sung et al. found that microurea serum Type 3 and Ureaplasma urealyticum serotype 10 are mostly found in the respiratory secretions of infants with severe BPD and death [4], and Naessens et al. proposed that Ureaplasma urealyticum serotype 4 is often found in pregnant women who have repeated abortions The above findings [6] show that the genotyping and discrimination of serotypes of uroplasma are helpful to clarify their pathogenic mechanism and epidemiological studies.

Uroplasma detection is more commonly used in women with urogenital tract infections and neonatal respiratory tract infections. The detection methods include traditional culture methods, polymerase chain reaction (PCR), real-time quantitative polymerase chain reaction (real-time PCR), etc. The traditional cultivation method is time-consuming, specific and has low sensitivity.

Real-time quantitative polymerase chain reaction mainly uses fluorescent dye and polymerase chain reaction to monitor the amount of reaction products in real time. The polymerase chain reaction uses DNA polymerase (such as Taq polymerase) to extend the area where specific primers are bound to synthesize new Double-stranded DNA. The fluorescent dye can be used with Taqman probe or SYBR Green I, etc. The use of SYBR Green I has the advantages of fast, easy operation and good sensitivity [9].

At present, many clinics and hospitals provide relevant services by real-time quantitative polymerase chain reaction method. The urease gene fragment can be used to design primers for the detection target for the detection of microureaplasma and ureaplasma The limits are 10 ⁷ and 10 ⁴ copies/mL, respectively.

Chinese patent CN102094072 A discloses a PCR kit for detecting ureaplasma urealyticum infection by SYBR Green method. The Chinese patent CN10149726 B discloses a kit for rapid and simultaneous detection of Chlamydia trachomatis, Ureaplasma minor and Ureaplasma urealyticum.

There is still a need for nucleic acid detection methods that can specifically detect microureaplasma or ureaplasma urealyticum of a specific serotype.

In one aspect, the present invention provides a primer pair for detecting Ureaplasma urealyticum serovar 10 (UU-10) whose DNA sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2. Show. The primer pair of the present invention can achieve rapid, stable, sensitive, and can accurately quantify and identify the specific serotype (UU-10) nucleic acid diagnosis of uroplasma.

The primer pair of the present invention is composed of a forward primer and a reverse primer, the forward primer is 5'-GCTAGTATTATCGATGCTAA-3' (the sequence is shown in SEQ ID NO: 1), and the reverse primer is 5'-CTCGATCAAACAAGTCAA -3' (sequence is shown in SEQ ID NO: 2).

The primer pair of the present invention can be used for PCR or RT-PCR. In a preferred embodiment of the present invention, the primer pair is used for RT-PCR in combination with SYBR Green I stain.

In another aspect, the present invention provides a kit for PCR detection of UU-10, which includes the primer pair as described above. In some embodiments of the present invention, the kit further includes a DNA-binding dye, for example, a SYBR Green I dye.

It should be understood that both the previous general description and the following detailed description are only exemplary and explanatory and do not limit the invention.

The following detailed description is only exemplary and explanatory, rather than limiting the present invention.

The invention provides a primer pair for detecting Ureaplasma urealyticum serovar 10 (UU-10), which is composed of a forward primer and a reverse primer, the forward primer is 5'-GCTAGTATTATCGATGCTAA-3' (sequence is shown in SEQ ID NO: 1), the reverse primer is 5'-CTCGATCAAACAAGTCAA-3' (sequence is shown in SEQ ID NO: 2).

The primer pair of the present invention can be used for PCR or RT-PCR. In a preferred embodiment of the present invention, the primer pair is used for RT-PCR analysis in conjunction with the SYBR Green I PCR reaction reagent, which has the advantage of being able to accurately quantify and specifically distinguish UU-10, and has a wide range of accurate quantification (10 ⁵ -10 ¹⁰ replicas/mL), high sensitivity (detection limit is 10 ⁴ replicas/mL).

In another aspect, the present invention provides a kit for PCR detection of UU-10, which includes the primer pair as described above. According to some embodiments of the present invention, the kit further includes a DNA binding dye. The DNA binding dyes include but are not limited to: SYBR Green I, SYBR Green II, GelStar and SYBR Gold dyes, or derivatives thereof.

In a preferred embodiment of the present invention, the PCR is RT-PCR. In a specific embodiment, the DNA binding stain is a SYBR Green I stain.

The primer pair or set of the present invention can achieve rapid, stable, sensitive, and can accurately quantify and identify the specific serotype (UU-10) nucleic acid diagnosis of uroplasma. In addition, the primer pair or kit of the present invention can be used in the risk assessment test for UU-10 infection and diseases caused by UU-10 infection, including but not limited to bronchopulmonary dysplasia in children , BPD) and its severity, meningitis, brain defect diseases...etc.

The invention is further illustrated by the following examples, which are provided for illustrative purposes and not to limit the invention.

The specific experimental conditions and methods are not indicated in the following examples, usually according to conventional conditions, such as molecular cloning edited by Sambrook et al.: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or reference reagents The conditions and procedures recommended by the manufacturer.

Examples

Examples 1 : Ureaplasma urealyticum serotype 10 Quantitative range and sensitivity analysis

Ureaplasma urealyticum serotype 10 (UU-10) positive plastid template was serially diluted 10 times to 10 ¹¹ copies/mL, 10 ¹⁰ copies/mL, 10 ⁹ copies/mL, 10 ⁸ copies/ mL, 10 ⁷ copy bodies / mL, 10 ⁶ copies of / mL, 10 ⁵ copies of / mL, 10 ⁴ copies of / mL and a negative control group (non-template control, NTC) . The UU-10 positive plastid template was prepared by inserting a specific 175 bp sequence of UU-10 strain (American Standard Strain Conservation Center ATCC No. 33699) (the 367945-368119 bp of NCBI sequence number NC_011374.1) into pUC57 plastid to prepare get.

Take 5.0 μL of each concentration in the negative control group and UU-10 positive plastid template, add polymerase reaction solution (2X, including SYBR Green I stain) 10.0 μL, the forward primer of SEQ ID NO: 1 1.0 μL (final concentration 0.5 μM), the reverse primer of SEQ ID NO: 2 1.0 μL (final concentration 0.5 μM), then adjust the total reaction volume of each reaction tube to 20 μL with double distilled water (ddH ₂ O), and perform three reaction tubes at the same concentration Repeated test.

The detection was carried out in a real-time quantitative polymerase reaction instrument. The instrument conditions were set as follows: a preparatory program for one cycle at 50 ^o C, 2 minutes, 95 ^o C, and 10 minutes, followed by 95 ^o C, 15 seconds, and 60 ^o C 、Perform 40 cycles of amplification procedure in 1 minute condition, last for 15 seconds at 95 ^o C and 1 minute at 60 ^o C, and rise to 95 ^o C at a rate of 0.15 ^o C per second for melting curve ( melting curve) test.

After the detection, the real-time quantitative polymerase reactor is used to analyze the number of cycles (Ct) obtained after the samples are defined by the threshold. The average of triplicate results is: 10 ¹¹ replicas/mL, 10 ¹⁰ replicas/mL, 10 ⁹ replicas/mL, 10 ⁸ replicas/mL, 10 ⁷ replicas/mL, 10 ⁶ replicas/mL, 10 ⁵ replisome / Ct values mL and ¹⁰⁴ copies of / mL of 9.00,13.62,16.88,20.30,23.52,26.62,29.87 and 27.88, respectively, the negative control group no amplification curve is generated. See Figure 1 .

The concentration of UU-10 positive plastid template was 10 ¹¹ replicas/mL, 10 ¹⁰ replicas/mL, 10 ⁹ replicas/mL, 10 ⁸ replicas/mL, 10 ⁷ replicas/mL, 10 ⁶ replicas/mL , copies of ¹⁰⁵ / mL and the Ct value corresponding to the regression analysis, R ² is 0.9998, as shown in FIG. 2, this range of concentrations may be displayed accurate quantification.

Repeat this operation three times, the PCR efficiency is between 90-105%, the linear regression R ² is higher than 0.99, the quantitative range is 10 ¹⁰ to 10 ⁵ replicas/mL, and the sensitivity can be 10 ⁴ replicas/mL. The accuracy of three independent test rooms (inter-assay) and the same test three replicates (intra-assay) (coefficient of variation = standard deviation / average x100%) is less than 15%, indicating that the test results between different batches are stable and reproducible high, see below table 1.

Table 1. Results of three independent tests

Examples 2 : Correct UU-10 Specific analysis

Take 5.0 μL of UU-10 positive plastid template as described in Example 1, add polymerase reaction solution (2X, including SYBR Green I stain) 10.0 μL, forward primer 1.0 μL (final concentration 0.5 μM), reverse Primer 1.0 μL (final concentration 0.5 μM), and then adjust the total volume of each reaction tube to 20 μL with double-distilled water (ddH ₂ O). Repeat the test with three reaction tubes at the same concentration.

The detection was carried out in a real-time quantitative polymerase reactor. The instrument conditions were set as follows: a preparatory procedure for one cycle at 50 ^o C, 2 minutes, 95 ^o C, and 10 minutes, followed by 95 ^o C, 15 seconds, and 60 ^o C 、Perform 40 cycles of amplification procedure in 1 minute condition, last for 15 seconds at 95 ^o C and 1 minute at 60 ^o C, and rise to 95 ^o C at a rate of 0.15 ^o C per second for melting curve ( melting curve) test.

After the detection, in order to match the real time quantitative polymerase reaction apparatus of FIG melting curve analysis software, as shown in FIG. 3, the result is a single melting curve, represents only a single primer amplification products having specific.

Examples 3 :Correct UU-10 Specific computer analysis

The present invention is related to the front case and the primers and probes to BLAST (Basic Local Alignment Search Tool) analysis can be provided to detect the urinary NCBI slurry serotype, the results in Table 2 below. From the results, it can be seen that the primer pair of the present invention can specifically identify UU-10 without identifying other serotypes of uroplasma.

*表示經BLAST分析可偵測之血清型。Table 2. Comparison of the serotypes of uroplasma detectable by the primer of the present invention and the primer or probe of the previous case

* Indicates the serotype detectable by BLAST analysis.

Those skilled in the art should understand that the above-described embodiments can be changed without departing from the broad concept of the invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by the scope of the patent application.

References: [1] Viscardi, Rose M. "Ureaplasma species: role in diseases of prematurity." Clinics in perinatology 37.2 (2010): 393-409. [2] Paralanov, V., et al. "Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains." BMC microbiology 12.1 (2012): 88. [3] Kallapur, Suhas G., Boris W. Kramer, and Alan H. Jobe. "Ureaplasma and BPD." Seminars in perinatology. Vol. 37. No. 2. WB Saunders, 2013. [4] Sung, TJ, et al. "Frequency of Ureaplasma serovars in respiratory secretions of preterm infants at risk for bronchopulmonary dsyplasia." The Pediatric infectious disease journal 30.5 (2011): 379 . [5] Kong, F., et al. "Species identification and subtyping of Ureaplasma parvum and Ureaplasma urealyticum using PCR-based assays." Journal of clinical microbiology 38.3 (2000): 1175-1179. [6] Naessens, A. , et al. "Serotypes of Ureaplasma urealyticum isolated from normal pregnant women and patients with pregnancy complications." Journal of clinical microbiology 26.2 (1988): 319-322. [7] Quinn, PA, et al. "Serologic evidence of Ureaplasma urealyticum infection in women with spontaneous pregnancy loss." American journal of obstetrics and gynecology 145.2 (1983): 245-250. [8] Zhang, Y ., et al., “Ureaplasma urealyticum serotypes distribution and drug resistance.” Chinese journal of zoonoses 32.1 (2016): 45-50. [9] Ponchel, F., et al. "Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions." BMC biotechnology 3.1 (2003): 18.

no

It is better to understand the present invention as described above and the following detailed description in conjunction with the reading of the drawings. The drawing shows the presently preferred embodiment.

Figure 1 shows the amplification plots of UU-10 positive plastid templates and negative control groups at different concentrations (10 ¹¹ -10 ⁴ replicas/mL).

Figure 2 shows the linear regression of UU-10 positive plastid template at 10 ¹⁰ -10 ⁵ replica/mL concentration.

Figure 3 shows the melting curve plots of UU-10 positive plastid templates and UU-10 primers at different concentrations.

no

<110> Xuanjie Stem Cell Biotechnology Co., Ltd. <120> Primer pair and its kit for detecting Ureaplasma urealyticum serotype 10 <130> MRD0006TW <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 1 gctagtatta tcgatgctaa 20 <210> 2 <211> 18 <212> DNA <213> artificial sequence <220> <223> Reverse primer <400> 2 ctcgatcaaa caagtcaa 18

Claims (5)

A primer pair for detecting ureaplasma urealyticum serotype 10, whose DNA sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2. A kit for PCR detection of Ureaplasma urealyticum serotype 10, which includes the primer pair as described in claim 1. As in claim 2, the kit further includes a DNA binding dye. As in the set of claim 3, wherein the PCR is RT-PCR. The kit according to claim 4, wherein the DNA-binding dye is SYBR Green I dye.

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