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Primer set and kit for detecting Ureaplasma urealyticum through loop-mediated isothermal amplification method

A Ureaplasma urealyticum, ring-mediated isothermal technology, applied in the field of microbial detection, can solve the problems of high experimental conditions and technical requirements, difficult to carry out in primary hospitals, and inability to produce results on the same day, to avoid DNA contamination and shorten clinical trials. The detection time and the effect of improving the clinical detection rate

Inactive Publication Date: 2019-01-01
JINAN CENTER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, liquid culture method and PCR method are mostly used in clinical detection of Uu. Among them, the liquid culture method is easy to operate and does not require high experimental conditions, but this method takes a long time and cannot produce results on the same day, which brings great inconvenience to outpatients. ; PCR detection technology has high sensitivity and high specificity, but this method has high requirements for experimental conditions and inspection techniques, and it is difficult to carry out in primary hospitals

Method used

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  • Primer set and kit for detecting Ureaplasma urealyticum through loop-mediated isothermal amplification method
  • Primer set and kit for detecting Ureaplasma urealyticum through loop-mediated isothermal amplification method
  • Primer set and kit for detecting Ureaplasma urealyticum through loop-mediated isothermal amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Design and screening of primers

[0043] A primer set was designed according to the 16S ribosomal RNA gene sequence of Ureaplasma urealyticum (its nucleotide sequence is shown in SEQ ID NO: 6). Then, according to the principle of LAMP reaction, five sets of conventional primers (see Table 1) were synthesized for screening, and each set of primers included two outer primers (F3 and B3) and two inner primers (FIP and BIP). Through a large number of experiments, the optimal primer set 4 and primer set 5 were screened out by monitoring the reaction progress and results. In order to speed up the LAMP reaction speed, corresponding loop primers (see Table 2) were designed for primer set 4 and primer set 5 for further screening. Finally, the best primer set with high specificity and fast response (see Table 3) was screened out, including two outer primers (F3 and B3), two inner primers (FIP and BIP) and one loop primer (LB).

[0044] Table 1 screening primer set se...

Embodiment 2

[0053] Embodiment 2: the kit that detects Ureaplasma urealyticum

[0054] 1. The composition of the kit: the combination of primers and probes screened in Example 1, reaction buffer, Bst DNA polymerase, chromogenic solution, sterile ultrapure water; wherein, the composition and content of the reaction buffer are shown in Table 4.

[0055] Table 4 Reaction buffer composition and content

[0056]

[0057] The activity content of Bst DNA polymerase is: 160 units.

[0058] The chromogenic solution is manganese ion chelated calcein: 0.001 μmol.

[0059] Sterile ultrapure water: 3.5 μL.

[0060] The content of each primer is shown in Table 5.

[0061] Table 5 Content of each primer in 25 μL reaction system

[0062]

[0063] 2. Kit configuration

[0064] The kit contains 10 reaction tubes containing 23 μL reaction solution; 1 positive control tube containing 25 μL Uu DNA, 1 negative control tube containing 100 μL sterile ultrapure water; 30 suitable for 1-10 μL pipetting ...

Embodiment 3

[0083] Example 3: clinical sample application detection

[0084] The DNA of 30 clinical specimens suspected of Ureaplasma urealyticum infection was extracted, and the LAMP detection kit established by the present invention was compared with the most widely used liquid culture method in clinical practice. The kit identified 5 positive samples, and the coincidence rate with the culture identification result was 100%. With this kit, it only takes 1 hour from the receipt of the clinical sample to the result, while the traditional culture identification takes 48 hours to get the result. In contrast, the kit of the present invention is faster and easier, and has the same accuracy as the liquid culture method.

[0085] In this experiment, 5 primers were designed for 6 different regions on the 16S ribosomal RNA gene sequence of Ureaplasma urealyticum to detect clinical samples, which ensured the specificity and sensitivity of the LAMP reaction. In summary, the color-based LAMP prime...

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Abstract

The present invention discloses a primer set and a kit for detecting Ureaplasma urealyticum through a loop-mediated isothermal amplification (LAMP) method. According to the present invention, the primer set is designed for six different regions on the 16S ribosomal RNA gene sequence of Ureaplasma urealyticum, and comprises a pair of specific internal primers, a pair of specific outer primers and aloop primer; the kit comprises 10 reaction tubes containing 23 [mu]L of a reaction solution, 1 positive control tube containing 25 [mu]L of Uu DNA, and 1 negative control tube containing 100 [mu]L ofsterile ultrapure water; the primer set and the kit can quickly and sensitively detect Ureaplasma urealyticum, have characteristics of high specificity and easy operation, and are suitable for rapiddiagnosis and screening in hospitals at all levels.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a primer set and a kit for detecting Ureaplasma urealyticum by a loop-mediated isothermal amplification method. Background technique [0002] Ureaplasma urealyticum (Uu) belongs to the Mycoplasma family Ureaplasma genus, is an opportunistic pathogen that parasitizes the human genitourinary tract, can be transmitted through sexual contact, delivery through the birth canal, etc., is non-gonococcal One of the main pathogenic bacteria of urethritis. In recent years, a large number of studies have confirmed that Uu is related to various diseases such as urinary stones, bladder stones, prostatitis, orchitis, neonatal low birth weight, and neonatal pneumonia. Ureaplasma urealyticum infection has become a public health problem that has attracted much attention. one. Early diagnosis has far-reaching significance for the treatment of Ureaplasma urealyticum infection, ther...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/35
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 王庆西汪运山杨炜华伦恒忠
Owner JINAN CENTER HOSPITAL
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