Trichomonad nested PCR detection kit and preparation method

A technology for detection kits and detection methods, applied in the fields of biochemical equipment and methods, microorganism-based methods, DNA/RNA fragments, etc., can solve the problem of low detection rate and reliability, long detection kit cycle, time-consuming and laborious and other problems, to achieve the effect of strong specificity, convenient observation results, and improved sensitivity and accuracy

Active Publication Date: 2017-01-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main method for detecting Trichomonas murine is smear microscopy, which is time-consuming and laborious, with low detection rate and reliability
The development of a detection kit related to immunological technology takes a long time and costs a lot, so it is the first choice to develop a specific and sensitive PCR detection kit for Trichomonas murine from the perspective of molecular biology technology
Due to the complex composition of stool samples, which contain a large number of organisms, false positive results often occur when using ordinary PCR methods

Method used

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  • Trichomonad nested PCR detection kit and preparation method
  • Trichomonad nested PCR detection kit and preparation method
  • Trichomonad nested PCR detection kit and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Design of primers for Trichomonas mouse nested PCR detection kit

[0037] According to the partially conserved sequence of the 16S rRNA gene of Tritrimonas murine and Pentatrimonas human, specific diagnostic primers were designed with biological software as follows:

[0038] First-round PCR primer: F 1 : 5'-ACA CTT CGG TCA TAG ATT AA-3'

[0039] f 2 : 5'-AGG GAT TCC TGG TTC AT-3'

[0040] Second-round PCR primer: R 1 : 5'-AGT TTG CTC CCA TAT TGT TG-3'

[0041] R 2 : 5'-CAC GGA CCT GTT ATT GCT AC-3'

[0042] F in the above two pairs of primers 1 , F 2 For the first round of PCR primers, R 1 , R 2 Primers for the second round of PCR. Among them, the first round can amplify a 1362bp or 1360bp band when the concentration of genomic DNA is high, and the second round can amplify a 724bp or 727bp diagnostic gene fragment. The target band is bright and there is no non-specific band ( See attached figure 1 ). The purpose of sensitive and specific detection of t...

Embodiment 2

[0050] 1. Optimization of reaction conditions for Trichomonas murine nested PCR

[0051] The optimization process of specific nested PCR reaction conditions is:

[0052] Mg 2+ Optimization of Concentration: Setting Mg 2+ The concentration gradients were 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, 2.5 mmol / L, 3.0 mmol / L, 3.5 mmol / L, 4.0 mmol / L, and the results showed that the best Mg 2+ The concentration is 2.0mmol / L (see attached figure 2 ). Optimization of the first round of PCR annealing temperature: set the annealing temperature gradients to 44.0°C, 47.0°C, 50.0°C, 53.0°C, 56.0°C, 59.0°C, 62.0°C, 65.0°C, 68.0°C, 71.0°C, the results show the best annealing The temperature is 50.0°C (see attached image 3 ).

[0053] Optimization of the annealing temperature for the second round of PCR: set the annealing temperature gradients to 44.0°C, 47.0°C, 50.0°C, 53.0°C, 56.0°C, 59.0°C, 62.0°C, 65.0°C, 68.0°C, 71.0°C, and the results show the best annealing The temperature i...

Embodiment 3

[0061] 1. Stability and repeatability test of Trichomonas mouse nested PCR detection kit

[0062] Store the positive template, nested PCR reaction solution, primers and r Taq Polymerase (5U / μl) at -20°C, and store bromophenol blue and other items at room temperature. When the storage time was 1 month, 2 months, 3 months, 5 months and 6 months, the kit was taken out, and the stability of the kit was identified with known positive samples. Take 10 positive samples and use the kit to repeat the test 3 times, and make a negative control at the same time. The results show that there is a specific target band at the size of 724bp or 727bp, while the negative control does not have any amplification bands. Therefore, the stability and repeatability of the kit sex is good.

[0063] , Shelf life test of Trichomonas mouse nested PCR detection kit

[0064] Kits stored at -20°C for 1 month, 3 months, and 6 months were taken out and tested on known positive samples. According to the inst...

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Abstract

The invention discloses a trichomonad nested PCR detection kit and a preparation method. A primer is designed according to a part of conserved sequences of 16S rRNA genes of trichomonad and trichomonas hominis daraine, a specific nested PCR detection method is established, the sensitivity and accuracy of the detection method are improved by optimizing the concentration of the magnesium <2+> involved in PCR reaction, annealing temperature and other reaction conditions, and result is convenient and quick to observe after an amplification prdocut is subjected to 1.0% agarose gel electrophoresis. The trichomonad and the trichomonas hominis daraine can be rapidly detected only by using one nested PCR primer, and the trichomonad nested PCR detection kit has the advantages of being simple in operation, high in high sensitivity, good in specificity and the like.

Description

technical field [0001] The invention discloses a trichomonas murine nested PCR detection kit, which is mainly used for trichomonas murine ( Tritrichomonas muris ) or Trichomonas hominis ( Pentatrichomonas hominis ), the invention also provides the preparation method and usage of the kit, belonging to the technical field of parasite detection. Background technique [0002] Trichomonas ( Trichomoniasis ) is the phylum Protozoa ( Protozoa ) Flagellate ( Flagellata ) Kinoflagellates ( Zoomastigina) Trichomonas ( Trichomonadida ) is an opportunistic pathogenic parasite. Clinically, rodents, especially Trichomonas murine ( T. muris ) and Trichomonas hominis ( P. hominis ) is the most pathogenic, and is commonly found in the digestive tract of various animals, especially in the cecum of mice. [0003] Rats such as white mice and guinea pigs are often used as experimental animals and are very important experimental materials. According to the national standard for e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6848C12Q1/686C12Q1/6893C12Q2549/119C12Q2531/113C12Q2565/125
Inventor 李建华朱刚宫鹏涛张西臣杨举李赫李棕松尹继刚任文陟
Owner JILIN UNIV
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