Babesiidae protozoon detection primer and real-time fluorescent quantitative PCR (polymerase chain reaction) kit

A real-time fluorescence quantitative, Babesia technology, applied in the biological field, can solve the problems of unsatisfactory results of ordinary PCR, cumbersome Babesia detection, long detection cycle, etc., and achieves the effects of good detection effect, high degree of automation, and simple operation.

Active Publication Date: 2014-04-09
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional detection method of Babesia is carried out by blood smear staining and microscopic examination, but the results of staining and microscopic examination of pictures are affected by many factors,

Method used

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  • Babesiidae protozoon detection primer and real-time fluorescent quantitative PCR (polymerase chain reaction) kit
  • Babesiidae protozoon detection primer and real-time fluorescent quantitative PCR (polymerase chain reaction) kit
  • Babesiidae protozoon detection primer and real-time fluorescent quantitative PCR (polymerase chain reaction) kit

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Effect test

Embodiment 1

[0026] The design of embodiment 1 primer and probe

[0027] In the present invention, a pair of oligonucleotide sequences (primers) and an oligonucleotide sequence (probe) are designed by searching and comparing the 18S rRNA gene sequences in members of Babesia murine origin, and the designed primers can specifically The gene fragment of Babesia 18SrRNA was amplified, and the amplified product was 170bp.

[0028] The designed primer sequences are shown in SEQ ID No.1 and SEQ ID No.2:

[0029] Primer 1 (bc246f) SEQ ID No. 1: 5′-GGCGATGTATCATTCAAG-3′;

[0030] Primer 2 (bc415r) SEQ ID No. 2: 5′-GTCAGGATTGGGTAATTTG-3′;

[0031] The designed probe sequence is shown in SEQ ID No.3. When synthesizing the sequence, its 5' is modified with FAM and NONE groups, that is, bc394probe: 5'-FAM-CGCCTGCTGCCTTCCTTAGA-NONE-3', as a probe;

[0032] Primers and probes are entrusted to Synthetic Synthesis.

[0033] The nucleotide sequence of the amplified product is shown in SEQ ID No.4, and S...

Embodiment 2

[0034] Embodiment 2 Babesia real-time fluorescent quantitative PCR detection kit

[0035] The detection kit includes the following components:

[0036] Taq DNA Polymerase Mixture (2×);

[0037] ROX dye;

[0038] Primer 1: its nucleotide sequence is shown in SEQ ID No.1;

[0039] Primer 2: its nucleotide sequence is shown in SEQ ID No.2;

[0040] TaqMan probe: the nucleotide sequence is shown in SEQ ID No.3, and its 5' end is marked with a FAM group, and its 3' end is marked with a NONE non-fluorescent group;

[0041] Negative control: sterilized ultrapure water;

[0042] Positive control: plasmid DNA carrying an amplified product, the nucleotide sequence of which is shown in SEQ ID No.4.

[0043] Wherein, the Taq DNA polymerase mixture (2×) is a mixture of 2 times the concentration of Taq DNA polymerase, Buffer, and dNTP mixture used in the PCR reaction; the ROX dye is ROX Reference Dye II (50×); The above Taq DNA polymerase mixture (2×) can also be Premix Ex Taq (2×). ...

Embodiment 3

[0045] The detection sensitivity of embodiment 3 Babesia real-time fluorescent quantitative PCR kit

[0046] (1) Acquisition of Babesia positive plasmid DNA: Synthetic by Shanghai Sangon.

[0047] (2) Gradient dilution of positive plasmid: carry out 10-fold ratio dilution, that is, 10 1 ~10 -7 , of which 10 -7 The concentration of plasmid was 1.34 x 10 -7 ng / μL.

[0048] (3) Preparation of real-time fluorescent quantitative RT-PCR reaction system:

[0049]

[0050] (4) Real-time fluorescent quantitative PCR reaction program:

[0051]

[0052] (5) Collect fluorescence signal and detect Ct value.

[0053] Test results such as figure 1 (the abscissa represents the cycle number Cycle Number, and the ordinate represents the fluorescence intensity Fluorescence), the results show that the positive plasmid was diluted to 1.34 × 10 -7 The signal can still be detected after the concentration of ng / μL, indicating that the sensitivity of the real-time fluorescent quantitativ...

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Abstract

The invention relates to detection of a pair of babesiidae protozoon detection primer, and the upstream and downstream primer sequences are shown as SEQ ID No.1 and SEQ ID No.2. A real-time fluorescent PCR (polymerase chain reaction) kit for babesiidae protozoon detection comprises a Taq (thermu aquaticus) DNA polymerase mixed liquid, a ROX (roxithromycin) dye, a primer 1 with a nucleotide sequence shown as the SEQ ID No.1, a primer 2 with a nucleotide sequence shown as the SEQ ID No.2, a TaqMan probe with a nucleotide sequence shown as SEQ ID No.3, a negative control and a positive control. The provided real-time fluorescent quantitative PCR detection kit is convenient to use, is less in reagent use amount, greatly simplifies the operation process, reduces pollution in the operation process, is strong in detection effect specificity and is high in sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer and a real-time fluorescent quantitative PCR kit for detecting Babesia. Background technique [0002] Babesiosis is a general term for a class of blood protozoa diseases caused by various protozoa of the genus Piroplasma, Piroplasma, Babesi, parasitic on animals and humans. Studies have shown that the main pathogens of human babesiosis are Babesia divergens and Babeisia microti. After human infection, it can cause hemoglobinuria, jaundice, high fever, etc., and severe cases can lead to death. [0003] The source of infection of babesiosis is the animal host, and the tick is the vector. Babesia cannot survive without the tick and host. The number of rodents is large, the distribution is wide, and the activity site is consistent with the habitat of ticks, which often have tick parasitism. Epidemiological data show that most of the patients in the crowd develop ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/90
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 刘丽娟陈倩富英群
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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