CN-121204297-B - Mitochondrial characteristic sequence, specific primer, kit and method for identifying Boletus sinense

CN121204297BCN 121204297 BCN121204297 BCN 121204297BCN-121204297-B

Abstract

The invention provides a mitochondrial characteristic sequence, a specific primer, a kit and a method for identifying Boletus sinensis, which belong to the technical field of Boletus sinensis identification, wherein the mitochondrial characteristic sequence is shown as SEQ ID NO.1, the mitochondrial gene specific primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3. The kit comprises the mitochondrial gene specific primer and an internal reference primer. According to the invention, a specific primer pair is designed aiming at a specific region on the mitochondrial gene of the Boletus sinense, and the quick and accurate identification of the Boletus sinense is realized by combining with an internal reference primer.

Inventors

Niu yingfeng
LIU JIN
ZHANG CHUNXIA
YANG TIANWEI
LIU NI

Assignees

云南省热带作物科学研究所

Dates

Publication Date: 20260512
Application Date: 20251125

Claims (6)

1. A mitochondrial nucleotide fragment for identifying Boletus sinense, which is characterized in that the sequence of the mitochondrial nucleotide fragment is shown as SEQ ID NO. 1.
2. The mitochondrial gene specific primer for identifying the Boletus sinense (Hu) is characterized by comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
3. A kit for identifying bolete sinensis, comprising the mitochondrial gene specific primer and the internal reference primer of claim 2.
4. A kit according to claim 3, wherein the sequence of the internal reference primer is shown in SEQ ID No.4 and SEQ ID No. 5.
5. The kit according to claim 4, wherein the internal reference primer is used for amplifying a characteristic sequence of bolete 18S rDNA as shown in SEQ ID NO. 6.
6. A method for identifying bolete of chinese saprophytics, comprising the steps of: 1) Extracting genome DNA of a sample to be detected; 2) Respectively amplifying the obtained genome DNA extracted in the step 1) by using a mitochondrial gene specific primer and an internal reference primer in the kit of claim 3 to obtain an amplified product; 3) According to the amplified products, determining whether the sample to be tested is Boletus sinensis, wherein when the amplified products of the internal reference primers are only 562bp and the amplified products of the specific primers are only 267bp, the sample is identified as Boletus sinensis, and when the amplified products of the internal reference primers are only 562bp and the specific primers are not amplified products or the amplified products are not 267bp, the sample is identified as other species of Boletus sinensis.

Description

Mitochondrial characteristic sequence, specific primer, kit and method for identifying Boletus sinense Technical Field The invention belongs to the technical field of bolete identification, and particularly relates to a mitochondrial characteristic sequence, a specific primer, a kit and a method for identifying bolete of Chinese saprophytics. Background The bolete family is a large basidiomycete with important economic and ecological values in the fungus kingdom. Among them, bolete (Buchwaldoboletus xylophilus) of chinese saprophytics, a rare saprophytics, is attracting attention due to its unique flavor and potential medicinal value. For the identification of Boletus sinesis, morphological identification is mainly relied on at present, and the identification is based on macroscopic features (such as pileus, stipe, fungus tube, fungus ring and the like) and microscopic features (such as spores, basidiomycetes, sacculus and the like) of fruiting bodies. However, this method has an inherent disadvantage in that the result of authentication is highly dependent on experience and subjective judgment of the authenticator, and erroneous judgment is easily generated. The fruiting body of fungi is easily affected by factors such as growth environment, climate, maturity and the like, so that the same species shows polymorphism, and different species may show similarity. Depending on the whole fruiting body, the method must rely on the fruiting body which is structurally complete and mature, and can hardly be identified for mycelium, dried specimens, processed products (such as fungus soup, powder) or rotted samples. Another method is tissue isolation culture identification, which attempts to perform pure culture by isolating mycelium and then observe colony morphology. However, for the saprophytic bacteria such as the bolete sinensis and the like which grow slowly and have strict requirements on culture conditions, the success rate is extremely low, the mycelium separation is extremely difficult, the culture period is long, and the requirement of rapid identification cannot be met. DNA barcode technology is the predominant method of fungal molecular identification, where the Internal Transcribed Spacer (ITS) region in the ribosome is recommended as the standard barcode for fungal identification. Although ITS sequence has better discrimination capability in most fungi, for the similar species of bolete of Chinese saprophyticus, the problem of insufficient variation or multicopy variation of ITS sequence may exist, resulting in insufficient resolution and difficulty in realizing accurate discrimination at the species level. Recent mitochondrial genes have evolved to be preferred targets for specific identification of fungal species by virtue of unique advantages. The mitochondrial gene has the characteristics of higher copy number in fungi, moderate evolution rate, obvious sequence difference among species, strong genetic stability, difficult interference by nuclear genes and the like. However, for Boletus sinense, there is no method for identifying mitochondrial genes. Disclosure of Invention Therefore, the invention aims to provide a mitochondrial characteristic sequence, a specific primer, a kit and a method for identifying the Boletus sinense, and the quick and accurate identification of the Boletus sinense is realized by designing a specific primer pair aiming at a specific region on a mitochondrial gene of the Boletus sinense. The invention provides a mitochondrial characteristic sequence for identifying Boletus sinense, which is shown as SEQ ID NO. 1. The invention provides a mitochondrial gene specific primer for identifying Boletus sinense, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3. The invention provides a kit for identifying Boletus sinense, which comprises a mitochondrial gene specific primer and an internal reference primer. Preferably, the sequences of the internal reference primers are shown as SEQ ID NO.4 and SEQ ID NO. 5. Preferably, the internal reference primer is used for amplifying the characteristic sequence of bolete 18S rDNA shown as SEQ ID NO. 6. The invention provides a method for identifying Boletus sinense, which comprises the following steps: 1) Extracting genome DNA of a sample to be detected; 2) Taking the genome DNA obtained by the extraction in the step 1) as a template, and respectively amplifying by using a mitochondrial gene specific primer and an internal reference primer in the kit to obtain an amplified product; 3) According to the amplified products, determining whether the sample to be tested is Boletus sinensis, wherein when the amplified products of the internal reference primers are only 562bp and the amplified products of the specific primers are only 267bp, the sample is identified as Boletus sinensis