Method for the detection of nucleic acid synthesis and/or amplification
A technology of target nucleic acid sequence and chelating agent, which is applied in the field of detection of nucleic acid synthesis and/or amplification, which can solve the problems of difficulty in observing color change and unskilled evaluation of observers, so as to reduce costs and improve the effect of chromaticity detection
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Embodiment 1
[0179] To follow the amplification reaction of methods known in the art, hydroxynaphthol blue was used in the LAMP method (Goto M et al., Biotechnology, 2009; Ma XJ et al., Virology, 2010, Cardoso TC et al., Molecular Cell Probes, 2010). When the applicant implemented the method in the prior art, a blue reaction mixture was obtained before the LAMP amplification. As the target nucleic acid amplification reaction progressed, the reaction mixture was not always observed to change color. This is because, using the method of the prior art, the amplification reaction solution before nucleic acid amplification is dark blue, and it is light blue at the end of possible amplification of the target nucleic acid, which is difficult to clearly distinguish from the color of the starting mixture. . figure 1 A shows positive and negative samples when the method of Goto et al. We suggest that a positive sample provides information about where in the sample the nucleic acid was amplified, th...
Embodiment 2
[0200] In order to prove that sodium citrate does not inhibit the LAMP amplification reaction, the applicant makes a positive control containing (Mycoplasma haemofelis), Bartonella (Candidatus Mycoplasma haemominutum), Bartonella (Candidatus Mycoplasma turicensis), Samples of Listeria monocytogenes, Leptospira interrogans, Pseudomonas fluorescens, Parvovirus were tested with and without lemon Sodium acid LAMP amplification reaction.
[0201] Specifically, 4 samples were prepared containing 4 mM and 6 mM magnesium and with and without 1.5 mM sodium citrate, respectively.
[0202] Specifically, these samples are:
[0203] Sample 1: LAMP reaction mixture containing 4 mM magnesium;
[0204] Sample 2: LAMP reaction mixture containing 4mM magnesium, 1.5mM sodium citrate;
[0205] Sample 3: LAMP reaction mix containing 6 mM magnesium;
[0206] Sample 4: LAMP reaction mixture containing 6mM magnesium, 1.5mM sodium citrate;
[0207] The LAMP lasted for 70 minutes at 65° C., and th...
Embodiment 3
[0212] Detection of Mycoplasma Contamination
[0213] In order to verify the effectiveness of the present invention, the DNA sample extracted from cat whole blood has been amplified by the method of the present invention to verify whether it is polluted by following pathogens: feline blood mycoplasma (Mycoplasma haemofelis), blood Bartonella (Candidatus Mycoplasma haemominutum) and Candidatus Mycoplasmaturicensis.
[0214] via commercial kit (Macherey-Nagel) extracted DNA from blood samples.
[0215] It has been demonstrated that possible contamination of samples can be detected by the method of the invention as well as by using PCR.
[0216] 20 samples were validated and the results obtained are as follows:
[0217] The results obtained by PCR:
[0218] - Candidatus Mycoplasma haemominutum: 9 positive samples (2 positive samples were also Mycoplasma haemofelis and 2 samples were also Candidatus Mycoplasma turicensis);
[0219] - Candidatus Mycoplasma turicensis: 2 posit...
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