Primer for detecting panda-derived babesia as well as kit and detection method thereof

A technology for source Babesia and Babesia, applied in the field of biology, can solve the problems of low sensitivity, unfavorable early diagnosis of giant panda-derived Babesia, and inability to accurately respond, so as to improve sensitivity and accuracy, significantly Sensitivity and practicality, and the effect of improving detection sensitivity

Active Publication Date: 2021-08-06
CHENGDU RES BASE OF GIANT PANDA BREEDING
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the lack of specific primers for Babesia from giant pandas significantly affects the detection rate of Babesia from giant pandas. At present, it is urgent to explore a specific primer and detection method for the detection of Babesia from giant pandas
[0003] For this disease, the most basic detection method is the blood smear microscopy method, which has a low detection rate and cannot distinguish the insect species, and cannot achieve early diagnosis. It is currently used as an auxiliary detection method in the acute onset stage; clinically Serological detection is often used for Babesia epidemiological research, but since there is no antibody against giant pandas on the market, the antibodies used in the detection have large species differences, which cannot accurately reflect the giant panda's infection with Babesia Therefore, in laboratory diagnosis, molecular diagnostic techniques are most commonly used, among which the common PCR detection technique is the most commonly used, but this method will have the problem of low sensitivity when detecting Babesia giant pandas , when the amount of worms carried by giant pandas is low, there will be problems such as missed detection, which is not conducive to the early diagnosis of Babesia from giant pandas

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer for detecting panda-derived babesia as well as kit and detection method thereof
  • Primer for detecting panda-derived babesia as well as kit and detection method thereof
  • Primer for detecting panda-derived babesia as well as kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Giant panda source Babesia nested PCR detection kit preparation

[0040] 1. Primer design and synthesis

[0041] According to previous studies, the Babesia was analyzed from the phylogenetic tree ( Figure 7 ), which is considered to be a new species of Babesia, which is significantly different from the main Babesia pathogens prevalent in China, and the existing primers are not suitable for the detection of Babesia from giant pandas. Moreover, the blood samples of giant pandas are more precious, and the amount obtained is less. At the same time, it is necessary to further improve the sensitivity for the detection of the recessive infection of Babesia in giant pandas, so that it can be applied to the detection of Babesia in giant pandas. Therefore, in the hypervariable region of the 18S rRNA sequence of the giant panda-derived Babesia, this application selected the specific sequence of the giant panda-derived Babesia, designed 2 pairs of specific primers fo...

Embodiment 2

[0051] Example 2 Sensitivity Detection

[0052] 1. It is 1ng / μL to measure the nucleic acid concentration of the positive plasmid in Example 1 with a nucleic acid protein analyzer (Nanodrop 2000). The positive plasmid obtained in Example 1 is diluted 10 times, and 2 μL of each dilution plasmid is selected as As a template, a total of 11 concentration gradients (1ng / μL-0.1ag / μL), using the commonly used primers BJ1 / BJ2 for Babesia detection (BJ1: 5'-GTCTTGTAATTGGAATGATGG-3'; BJ2: 5'-TAGTTTATGGTTAGGACTACG-3') Perform conventional PCR amplification. figure 2 It is the amplification result of routine PCR sensitivity detection of Babesia giant panda. A single target band (500bp) can be amplified in lanes 1-8, indicating that the minimum concentration of this detection is 10ag / μL.

[0053] 2. The positive plasmid (nucleic acid concentration is 1ng / μL) was diluted 10 times until it was diluted to the 11th concentration (0.1ag / μL), and 2μL of each dilution plasmid was selected as a...

Embodiment 3

[0055] Embodiment 3 specific detection

[0056] According to literature reports, the blood parasites currently infecting giant pandas include hepatic clusterworms, toxoplasma gondii, and Babesia in the invention. The detection samples in the present invention are mainly giant panda blood samples. In order to eliminate the detection method, hepatic clusterworms and The possibility of Toxoplasma gondii indicates the specificity of this detection method, and the specificity test of this method is hereby carried out.

[0057] Toxoplasma gondii (Genbank: L24381.1) and hepatic clusterworm plasmid (Genbank: AY150067.2) were used to verify the specificity of the primers and kit of the present invention. Select 2 μL of each plasmid as a template, use primers GPB18-F1 and GPB18-R1 for the first round of PCR amplification, use the 10-fold dilution of the first round of PCR product DNA as a template, and use primers GPB18-F2 and GPB18-R2 for amplification. Second round of PCR amplificati...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a primer for detecting panda source babesia as well as a kit and a detection method thereof. The primers comprise outer primers as shown in SEQ ID NO. 1 and SEQ ID NO. 2, and inner primers as shown in SEQ ID NO. 3 and SEQ ID NO. 4. The detection method comprises the following steps: (1) extracting panda blood DNA for later use; (2) taking the DNA obtained in the step (1) as a template, and carrying out a first round of PCR by adopting an outer primer pair; and (3) carrying out a second round of PCR by taking the first round of PCR product as a template and adopting the inner primer pair, and then carrying out gel electrophoresis and judging whether the babesia is contained or not. Four specific primers are designed aiming at a specific region taking 18S rRNA of the panda-derived babesia as a target gene, so that the problem of lack of a molecular detection method aiming at the panda-derived babesia is solved, and a specific and sensitive early diagnosis method for the panda-derived babesia is realized.

Description

technical field [0001] The invention belongs to the technical field of biology, and in particular relates to a primer for detecting Babesia derived from giant pandas, a kit and a detection method thereof. Background technique [0002] Babesia is an apicomplexan protozoa that parasitizes in the erythrocytes of vertebrates. Ticks are the main transmission medium, and erythrocytes are the only host cells that can infect. Babesiosis caused by Babesia infection is a worldwide blood protozoan disease, and its main clinical symptoms are high fever, anemia, jaundice and hemoglobinuria. More than 100 species of Babesia have been identified so far. The disease not only causes huge losses to animal husbandry and the national economy, but also seriously endangers the health of wild animals and population protection. It can harm animals such as canines, cats, raccoons, deer and primates, and some species can also be infected by humans. Report. Babesia from giant pandas (GenBank: MT256...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/6893C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/6893C12Q1/6848C12Q2600/166C12Q2531/113C12Q2549/119C12Q2545/113C12Q2565/125Y02A50/30
Inventor 岳婵娟刘颂蕊李运莉张东升齐敦武马锐邓泽帅苏小艳李林燕霞侯蓉
Owner CHENGDU RES BASE OF GIANT PANDA BREEDING
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap