Pythium splendens braun fluorescence quantitative PCR detection reagent and detection kit and application

Abstract

The invention discloses a pythium splendens braun fluorescence quantitative PCR detection reagent, a detection kit and an application; the detection reagent comprises a pair of specific primers with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a specific fluorescence probe with a sequence as shown in SEQ ID NO: 3; an amplification target fragment has a length of 94 bp, and the nucleotide sequence of the amplification target fragment is shown in SEQ ID NO: 4; the detection kit comprises components of a CTAB extract, a PCR buffer containing the primers and the probe, TaqDNA polymerase, positive control liquid, and quantitative standard liquid; the detection method comprises the extraction of total RNA and the fluorescence PCR reaction; detection performed by using the pythium splendens braun fluorescence quantitative PCR detection reagent overcomes disadvantages of time consumption, easy pollution, and electrophoresis detection necessity after amplification of routine PCR, can realize rapid and accurate qualitative and quantitative detection of pythium splendens braun in a sample, and has the advantages of simplicity, easy operations, intuitive results, high sensitivity, good repeatability, and the like.

Description

technical field [0001] The invention relates to a detection technology of pathogenic fungi, in particular to a fluorescent quantitative PCR detection reagent, a detection kit and an application of oil palm damping-off pathogens. Background technique [0002] oil palm damping-off fungus ( Pythium splendens ), also known as Pythium ornate, is an important disease on oil palm. Elaeis guineensis ), can also infect maize ( Zeamays ),sunflower( Helianthus annuus ),barley( Hordeum vulgare ),cucumber( Cucumissativus ), sweet potato ( Ipomoeabatatas ),wheat( Triticum aestivum ),broad bean( Viciafaba ),cowpea( Vignasinensis ), capsicum ( Capsicum spp.), tobacco ( Nautilocalyxlynchei ), Pelargonium ( Pelargonium spp.), Begonia ( Begonia spp.) and other more than 250 species of plants, causing symptoms such as dehydration and wilting of the whole plant of the host seedlings, browning and rot of the roots, stem bases, incisions, and shedding of the cortex. At...

AI Technical Summary

Problems solved by technology

Biological identification methods are labor-intensive and time-consuming, and some morphological features are unstable and may degenerate or disappear, and oil palm damping-off bacteria are heterothallic, and monosexual culture cannot produce sexual organs, which increases the difficulty of identification; Wang et al. The PCR sensitivity of the specific primers developed is not very high, and it is impossible to detect DNA samples with low content; ribosomal DNA sequence analysis, although the amount of biological information obtained is large, but the experimental operation is cumbersome and the detection cycle is long; RFLP can save a lot of time in sequence analysis. The procedure is cumbersome, but multiple enzyme digestions are required, which is time-consuming and labor-intensive, and the above molecular detection methods must be subjected to PCR post-processing such as electrophoresis, and contact with toxic reagent ethidium bromide, which is also prone to cross-contamination and false positive results