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Fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dirofilaria immitis

A fluorescent quantitative, heartworm technology, applied in the field of probes and kits, fluorescent quantitative PCR primers, can solve the problems of long infection incubation period, insufficient sensitivity, high false negative rate, etc.

Inactive Publication Date: 2014-04-09
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of the existing PCR method is not enough for rapid, effective and accurate molecular detection of Dirofilaria immigatus.
What is especially important is that the incubation period of heartworm disease infection is long and most of them are recessive infections, while the existing market monitoring methods have a high false negative rate due to insufficient sensitivity

Method used

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  • Fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dirofilaria immitis
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dirofilaria immitis
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dirofilaria immitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Detection of 40 dog whole blood nucleic acids from Costa Rica

[0043] 40 parts of dog whole blood collected from Costa Rica, 200 microliters of whole blood was extracted by commercial nucleic acid extraction kit for each part, and nucleic acid was eluted with 200 microliters of eluent. Use the PCR detection system that the present invention establishes, detect these 40 dog whole blood nucleic acids, according to the amplification curve and melting curve of real-time fluorescence quantitative PCR ( figure 2 ; image 3 ) judgment, 9 of the 40 samples were positive. The positive rate reached 22.5%. The PCR amplification products of 9 positive samples were purified by commercial kits and sent to a specialized sequencing company for sequencing. , compare the sequencing results on NCBI. All 9 positive samples were found to be Dirofilaria immigatus. Each PCR detection reaction is accompanied by a sequence-confirmed standard strain as a positive control, and a...

Embodiment 2

[0044] Example 2: Detection of 39 dog whole blood nucleic acids from Nicaragua

[0045] 39 parts of dog whole blood collected from Nicaragua, each part gets 200 microliters of whole blood and extracts through commercial nucleic acid extraction kit, uses the PCR detection system that the present invention establishes, judges according to the amplification curve of real-time fluorescence quantitative PCR, these 39 parts All samples were negative. Each PCR detection reaction is accompanied by a sequence-confirmed standard strain as a positive control, and a dilution as a negative control. Positive controls diluted in different times showed positive results, and negative controls showed negative results, indicating the correctness of the test results.

Embodiment 3

[0046] Example 3: Detection of 90 dog whole blood nucleic acids from three regions in China

[0047] 90 parts of dog whole blood collected from Guangzhou, Kunming and Zhengzhou in China (30 parts in each region), 200 microliters of whole blood was extracted from a commercial nucleic acid extraction kit for each part, and then eluted with 200 microliters The nucleic acid is eluted with liquid, which is used in the PCR detection system established in the present invention. According to the amplification curve of real-time fluorescence quantitative PCR, all 90 samples were negative. These dogs are all pet dogs who come to the animal hospital for treatment. The growth environment is relatively hygienic and nutritionally good, and many dogs will be vaccinated regularly, so they have less chance of being exposed to pathogens. Perhaps this is the reason for the all-negative results. This may be related to the differences in the geographical environment of different regions and the e...

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Abstract

The invention relates to a fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dirofilaria immitis. The sequences of the primer are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the probe are shown as SEQ ID NO.3 and SEQ ID NO.4. The primer and the probe can be adopted for detecting dirofilaria immitis through an FRET (fluorescence resonance energy transfer)-PCR technology, and can specifically amplify the nucleic acid of a unique pathogen dirofilaria immitis causing the dirofilaria immitis disease, without the amplification of nucleic acid of other similar filaria which can not cause dirofilaria immitis disease.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR primer, a probe and a kit for detecting Dirofilaria imimaginae. Background technique [0002] Dirofilaria immitis can cause blood parasitic nematode disease (Heartworm disease), which not only seriously affects the development of dog breeding, but also poses a serious threat to human health. The detection methods of Dirofilaria imimaginae mainly include: 1) Microfilariae inspection. Mainly through the blood test for microfilariae, it can be confirmed that there are adult parasites in dogs. However, since microfilariae cannot be detected in the blood in the early stage of heartworm infection, and 10%-67% of dogs naturally infected with heartworm are monosexually infected, microfilariae cannot be produced, resulting in This method is prone to false negatives. At the same time, the small number of parasitic adults or improper blood collection time and location will also affect the detection rate o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 王成明危蓝菁张继垒宋春莲B·卡腾巴克
Owner YANGZHOU UNIV
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