Method and kit for detection of schistosomiasis through polymerase chain reaction

A technology of schistosomiasis and reagent kit, applied in the field of diagnosing schistosomiasis

Inactive Publication Date: 2002-08-28
奥斯瓦尔多克鲁兹基金会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0016] Likewise, the search for rapid and effective methods for the detection of Schistosoma sp

Method used

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  • Method and kit for detection of schistosomiasis through polymerase chain reaction
  • Method and kit for detection of schistosomiasis through polymerase chain reaction
  • Method and kit for detection of schistosomiasis through polymerase chain reaction

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Embodiment 1

[0047] The present invention is described in detail by referring to the following examples. It must be understood that the present invention is not limited to these embodiments, but also includes changes and modifications within the functional scope of the invention. Example 1 Rupture Schistosoma monsonii eggs

[0048] Eggs were extracted from the liver of mice previously infected with 100 cercariae of Schistosoma monsonii and stored in 0.9% saline solution at -20°C until use (Pellegrino and Siqueira, 1956, Rev. Bras. Malar. 8:589) . To disrupt eggs, 10 μl of saline solution containing 100,000 eggs / ml was mixed with 90 μl of distilled water and the final solution was shaken for 5 minutes in a mechanical mixer. This mixture containing ruptured eggs was used directly for DNA extraction. Example 2 By the modified Steiner method (J.J.Steiner, C.J.Polkemba, R.G.Fjellstrom, and L.F.Elliott, 1995, a rapid single-tube genomic DNA extraction method for PCR and RAPD analysis, Nucleic...

Embodiment 4

[0051]Figure 2 shows the electrophoretic pattern obtained from the amplification of Schistosoma monsonii DNA and the maximum sensitivity achievable for the detection of this DNA. Lane M is the molecular weight standard; lanes 1-5 are 20, 10, 5, 1, and 0.5fg DNA in sequence. The PCR reaction was able to detect as little as 1 fg of Schistosoma monsonii DNA. Figure 3 shows the electrophoretic patterns obtained after amplifying DNA from five other Schistosoma species using the same primers under the same PCR conditions as used for Schistosoma monsonii. Lane M: molecular weight standard; Lane 1: Schistosoma monsonii; Lane 2: Schistosoma haematobium; Lane 3: Schistosoma japonicum (strain from the Philippines); Lane 4: Schistosoma japonicum (strain from Japan); Lane 5: Schistosoma sheep (S .matthei); Lane 6: Schistosoma bovis (S.bovis); Lane 7: S. leipperi; Lane 8: negative control. The PCR reaction was able to amplify DNA from all species of schistosomes examined. Figure 4 shows ...

Embodiment 5

[0054] Figure 6 shows the results of previous Kato-Katz assays for the amplification of S. monsonii DNA in feces of patients with various concentrations of the parasite. Lane M: molecular weight standard; Lane 1: positive control; Lane 2-9: human feces samples containing 0.96, 0.168, 432, 600, 96.912, and 72 eggs per gram in sequence. PCR results were consistent with those obtained by parasitological examination in all samples. Example 5 Detection of Schistosoma monsonii in patient serum

[0055] For 4 serum samples, 100 μl of each sample was used to purify DNA using a Glass-max(R) DNA separation spin column system (Life Technologies) according to the manufacturer's instructions. Then 2 µl of this DNA was used for PCR amplification under the same conditions as above. The human sera used were previously tested by the Kato-Katz method, 2 samples were positive and 2 samples were negative.

[0056] Figure 7 shows the amplification results. Lane M: molecular weight standard; La...

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Abstract

The object of the present invention is the detection of Schistosoma species in biological samples by PCR. The first embodiment of the present invention relates to a method for diagnosing schistosomiasis, that is, amplifying the DNA sequence of Schistosoma species by PCR, then separating the amplified products by electrophoresis, and finally detecting by appropriate techniques. In a second embodiment, the invention is directed to a diagnostic kit for the detection of schistosomiasis. The basic kit contains all the necessary reagents for PCR technology, that is, specific primers, nucleotides, and appropriate buffers for PCR amplification. The kit may optionally include Taq polymerase in sufficient quantity for amplification, standard DNA as a positive control for the reaction, buffer for preparing amplified material for electrophoresis, and a protocol and instruction manual for users.

Description

[0001] The present invention relates to methods and kits for diagnosing schistosomiasis in human samples by polymerase chain reaction (PCR). The method, based on the detection of parasite DNA, is especially useful in cases of low infection intensity, where the parasitological stool test (Kato-Katz) exhibits low sensitivity. Background of the invention [0002] Schistosomiasis is a disease caused by infection with parasites of the genus Schistosoma, the main infecting species being Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobiom. The disease has been documented for 4,000 years and still constitutes a major public health problem, affecting more than 200 million people in underdeveloped countries. Schistosoma monsonie is endemic in 54 countries and in South America, the Caribbean, Africa, and the Eastern Mediterranean. [0003] These worms (hermaphrodites) infect humans by penetrating the skin through cercariae (larval stage), whi...

Claims

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Application Information

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IPC IPC(8): G01N33/50C12N15/09C12Q1/68C12Q1/6888G01N27/447G01N33/58
CPCC12Q1/6888Y10S435/81
Inventor A·L·特勒斯拉贝罗E·迪阿斯奈托L·A·庞特斯
Owner 奥斯瓦尔多克鲁兹基金会
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