阅读说明：本技术 用于检测链球菌的方法、寡核苷酸和试剂盒 (Methods, oligonucleotides and kits for detecting streptococci ) 是由 张太松 刘学锋 朱丽敏 褚杰 何敏 于 2019-11-25 设计创作，主要内容包括：本发明公布了用于检测链球菌的寡核苷酸、试剂盒和方法,其都涉及用于检测链球菌属基因组中一个保守性区域的第一引物对和第一探针；用于检测B族链球菌基因组中第一个保守性区域的第二引物对和第二探针；用于检测B族链球菌基因组中不同于所述第一个保守性区域的第二个保守性区域的第三引物对和第三探针,采用荧光PCR技术,同时检测链球菌属及其成员B族链球菌,并且在检测B族链球菌时,同时扩增B族链球菌基因组中的两个不同的保守性区域,可以避免漏检。(The invention discloses oligonucleotides, kits and methods for detecting streptococcus, all involving a first primer pair and a first probe for detecting a conserved region in the streptococcus genome; a second primer pair and a second probe for detecting a first conserved region in the group B streptococcus genome; and a third primer pair and a third probe for detecting a second conserved region different from the first conserved region in the group B streptococcus genome are used for simultaneously detecting streptococcus and the member group B streptococcus thereof by adopting a fluorescent PCR (polymerase chain reaction) technology, and when detecting the group B streptococcus, simultaneously amplifying the two different conserved regions in the group B streptococcus genome so as to avoid omission.)

1. An oligonucleotide for detecting streptococcus, said oligonucleotide comprising (1) a first primer pair and a first probe for detecting a conserved region in the streptococcus genome; (2) a second primer pair and a second probe for detecting a first conserved region in the group B streptococcus genome; (3) a third primer pair and a third probe for detecting a second conserved region in the group B Streptococcus genome that is different from the first conserved region.

2. The oligonucleotide of claim 1, further comprising (4) a fourth primer pair and a fourth probe for detecting an internal standard gene.

3. The oligonucleotide of claim 1, wherein the conserved region of the streptococcus genome is from a 16S rRNA gene, a 23S rRNA gene, or a 16S-23S rDNA spacer.

4. The oligonucleotide of claim 1, wherein the first conserved region is from a Sip or Cfb gene.

5. The oligonucleotide of claim 1, wherein the second conserved region is from a Sip or Cfb gene.

6. The oligonucleotide of claim 1, wherein the base sequences of the first primer pair and the first probe are SEQ ID NOS: 1 to 3, respectively.

7. The oligonucleotide of claim 1, wherein the base sequences of the second primer pair and the second probe are SEQ ID NOS 4-6, respectively.

8. The oligonucleotide of claim 1, wherein the base sequences of the third primer pair and the third probe are SEQ ID NOS 7 to 9, respectively.

9. The oligonucleotide of claim 2, wherein the base sequences of the fourth primer pair and the fourth probe are SEQ ID NOS: 10 to 12, respectively.

10. The oligonucleotide of claim 1, wherein the fluorescent reporter and quencher groups of each probe are selected from any one of FAM-tamma/BHQ 1, VIC/HEX/JOE-TAMRA/BHQ1, ROX/Texas Red-BHQ2, and Cy5/LC Red640-BHQ2/BHQ 3.

11. A method of detecting streptococcus, the method comprising: (1) extracting DNA in a sample; (2) performing fluorescence PCR amplification on the DNA in the step (1) in a single tube in the presence of a group of primers and probes; (3) determining the presence or absence of group B streptococcus in the sample and, if not, the presence or absence of other streptococcus bacteria, wherein: the set of primers and probes comprises oligonucleotides according to any one of claims 1 to 10.

12. A kit for detecting streptococcus, said kit comprising a fluorescent PCR reaction solution, wherein said fluorescent PCR reaction solution comprises an oligonucleotide according to any one of claims 1 to 10.

13. The kit according to claim 12, further comprising a nucleic acid extract 1 and a nucleic acid extract 2, wherein the nucleic acid extract 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1-2% SDS, and the nucleic acid extract 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100.

Technical Field

The invention belongs to the fields of bioscience and biotechnology, and particularly relates to a method, oligonucleotide and kit for detecting streptococcus and group B streptococcus.

Background

Streptococcus (Streptococcus) is a common gram-positive bacterium in a large group of pyogenes. At present, the streptococcus is of 69 species and subspecies, widely distributed in nature, human and animal feces, and nasopharynx of healthy people, and most of them are not pathogenic. The diseases caused by streptococcus in human body mainly include various pyogenic inflammations, scarlet fever, neonatal septicemia, bacterial endocarditis, rheumatic fever, glomerulonephritis and other hypersensitivity diseases.

According to the haemolytic characteristics on blood agar medium, the genus Streptococcus can be divided into three distinct types: (1) alpha hemolytic streptococcus, also known as grass green streptococcus, has grass green hemolytic rings around colonies, usually inhabits the oropharyngeal cavity, respiratory tract and intestinal tract of a human, has weak pathogenicity and is mostly conditional pathogenic bacteria; (2) the beta hemolytic streptococcus generates strong hemolytic toxin, can enable colorless and transparent hemolytic rings with width of 2-4 mm to appear around colonies on a blood agar culture medium, has strong pathogenicity, and can cause human and animals to suffer from various diseases; (3) streptococcus C, insoluble blood, has no hemolytic ring around the colony, and is generally not pathogenic.

The streptococcus strains are divided into A, B, C, D groups of 20 groups according to the antigen structure of the streptococcus strains, and each group is divided into a plurality of subgroups according to the difference of surface protein antigens. About 90% of streptococcus strains pathogenic to humans belong to group a, which is occasionally seen in group B, C, D, G.

The diseases caused by streptococcus are characterized by complexity and diversity, on one hand, because the bacteria are of various types and have both invasiveness and toxin; on the other hand, tissues and organs of the human body are highly susceptible, and allergic reaction mechanisms are involved in the pathogenesis. Streptococcal invasive enzymes are produced by: hyaluronidase, streptokinase, and the like; the toxins produced were: streptolysin, rubella toxin. Allergic diseases such as rheumatic fever, acute glomerulonephritis, etc. can be caused by group A streptococcal infection.

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a common gram-positive Streptococcus known for its cell wall polysaccharide C species belonging to group B of the lansfield antigen structural classification. Group B streptococci are divided into 9 serotypes (types Ia, Ib, II to VIII) according to the antigenicity of capsular polysaccharides of the streptococci. Group B streptococcus is often present in rectum and vagina, and can cause infection of amniotic cavity of a parturient, premature rupture of fetal membranes, premature delivery of fetuses, abortion and the like. Statistically, about 10% to 30% of pregnant women are infected with group B streptococci, of which 40% to 70% are transmitted to the newborn during delivery. If the newborn carries the bacterium, about 1% to 3% of the newborn will develop early invasive infection, meningitis, pneumonia, septicemia, and 5% of them will cause death. Relevant researches show that the antibacterial drug can effectively prevent the occurrence of poor pregnancy fate such as premature rupture of fetal membranes of lying-in women by aiming at the use.

Prenatal screening for group B streptococci is of paramount importance, based on the adverse symptoms associated with infection with group B streptococci in pregnant women and newborns. In 2010, the U.S. disease prevention center designates 'guidance for prevention of group B streptococcus in perinatal period', and pregnant women are recommended to screen group B streptococcus 35-37 weeks of gestation.

In the past, for quite some time, GBS diagnosis has been achieved mainly by culturing maternal rectal or vaginal swabs, neonatal blood or cerebrospinal fluid, but conventional culture methods can achieve false negative rates of up to 50% in detecting streptococcal infection in pregnant women. The growth of other microorganisms can be inhibited by the use of selective media, which has been the gold standard for the determination of group B streptococcal infections. However, since the selective culture requires 72 hours for a negative result and at least 48 hours for a positive result, a method for rapidly detecting group B streptococci is required to make up for the disadvantage.

Due to the implementation of antibiotic prevention strategies for group B streptococci and the wide application of antibiotics, the traditional culture and serological detection methods are inevitable to cause the problem of insufficient sensitivity and the false negative of patients with mild infection and antibiotic treatment. The emergence and rapid development of molecular biology technology provides a new technical platform for rapid and sensitive diagnosis of group B streptococcus. There are mainly the following categories of methods for clinical diagnosis and/or strain study of group B streptococcus: molecular hybridization diagnosis, Polymerase Chain Reaction (PCR) and related technology diagnosis, molecular biology typing technology and the like.

Molecular hybridization diagnostics are accomplished by detecting group B streptococcal DNA or RNA with specific probes. In situ hybridization, Southern blot hybridization (Southern blot), and dot blot hybridization (dot blot) are common techniques. Foreign countries often use rRNA designed hybridization kits aiming at the group B streptococcus to detect the group B streptococcus infection, and the detection sensitivity of the kit in pregnant women is 94.17-100%, and the specificity is 96.19-99.15%. Recently, Fluorescence In Situ Hybridization (FISH) is reported to be used for detecting the bacteria carrying rate of the group B streptococcus of the pregnant women, and the FISH can diagnose 98.13 percent of bacteria carriers, while the standard culture method has a positive rate of only 64.14 percent. The FISH detection result is visual, but the test process is complicated, the related reagents are various, time and labor are wasted, the result needs to be interpreted by professionals with rich experience, and the result interpretation has larger subjectivity.

The development of Polymerase Chain Reaction (PCR) technology provides a powerful tool for rapid, sensitive, specific detection of pathogens in a variety of clinical specimens. Nested PCR detection of ribosomal operon genes has also been the subject of recent years, and this method detects group B streptococci by amplifying the ribosomal 16S rRNA gene or the 16S-23S spacer region. Its specificity is 87% -96%, but it has a certain false positive, probably due to the higher homology of ribosomal operon between bacterial species, such as 70% homology between the 16S-23S rDNA of S.difficile and B group streptococci. The determination of the whole genome sequence of the group B streptococcus provides more information for selecting other specific detection targets in the group B streptococcus except the ribosome operon. Group B streptococci were tested by the authors by designing primers for the scpB gene encoding C5a peptidase. Primers have also been designed for genes encoding the C.alpha.protein, the bc gene for the C.beta.protein, the bac gene for the C.alpha. -like proteins, alp2 and alp3 for the C.alpha. -like proteins, and the rib gene for the rib protein, and studies have been made on the gene distribution and variation of group B streptococcal surface proteins.

However, there is no prior art that detects streptococci simultaneously with group B streptococci, which indicates the presence of other streptococci even if group B streptococci are not present in the clinical specimen, which aids the clinician in further pathogen screening. In addition, the existing PCR technology is often used for detecting a single gene segment of group B streptococcus, which is easy to cause the problem of missed detection.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method, oligonucleotide and kit for detecting streptococcus, two groups of primers and probes are designed by selecting two different conserved regions (for example, one conserved region is from an Sip gene, and the other conserved region is from a Cfb gene) in a group B streptococcus genome, a group of primers and probes are designed as an internal control according to a conserved sequence (for example, a conserved sequence from a 16S rRNA gene, a 23S rRNA gene or a 16S-23S rDNA spacer region) in a streptococcus genome, and a human genome housekeeping gene beta-globin is introduced as an internal reference to establish a dual-quality control and dual-target verification system, so that a series of abnormal results encountered in the detection of clinical samples can be effectively avoided, and the problem of missed detection of the group B streptococcus can be solved.

In the present invention, x1, x2, x3 and x4 at the 5 'end of each probe are fluorescent reporter groups, and y1, y2, y3 and y4 at the 5' end of each probe are quencher groups which emit fluorescence or do not emit fluorescence.

The fluorescent reporter and quencher of the same probe of the present invention are given in the form of, for example, "FAM-TARMA/BHQ 1" or "VIC/HEX/JOE-TAMRA/BHQ 1", with the left of "-" representing the fluorescent reporter and the right of "-" representing the quencher. FAM-TARMA/BHQ1 shows that the fluorescence reporter of one probe is FAM, and the quenching group can be selected from TARMA or BHQ 1. VIC/HEX/JOE-TAMRA/BHQ1 shows that the fluorescence reporter gene of one probe can be selected from VIC, HEX or JOE, and the quenching group can be selected from TAMRA or BHQ 1.

The present invention provides an oligonucleotide for detecting streptococcus, which is characterized in that the oligonucleotide comprises (1) a first primer pair and a first probe for detecting a conserved region in a streptococcus genome; (2) a second primer pair and a second probe for detecting a first conserved region in the group B streptococcus genome; (3) a third primer pair and a third probe for detecting a second conserved region in the group B Streptococcus genome that is different from the first conserved region.

Further, the oligonucleotide further comprises (4) a fourth primer pair and a fourth probe for detecting an internal standard gene.

Further, the conserved region of the Streptococcus genome is from a 16S rRNA gene, a 23S rRNA gene, or a 16S-23S rDNA spacer.

Further, the first conserved region is from a Sip or Cfb gene.

Further, the second conserved region is from a Sip or Cfb gene.

Further, the base sequences of the first primer pair and the first probe are respectively SEQ ID NO. 1-3.

Further, the base sequences of the second primer pair and the second probe are respectively SEQ ID NO. 4-6.

Further, the base sequences of the third primer pair and the third probe are respectively SEQ ID NO. 7-9.

Further, the base sequences of the fourth primer pair and the fourth probe are respectively SEQ ID NO 10-12.

Further, the fluorescence reporter gene and the quenching group of each probe are selected from any one of FAM-TARMA/BHQ1, VIC/HEX/JOE-TAMRA/BHQ1, ROX/Texas Red-BHQ2 and Cy5/LC Red640-BHQ2/BHQ 3.

The invention also provides a method for detecting streptococcus, which comprises the following steps: (1) extracting DNA in a sample; (2) performing fluorescence PCR amplification on the DNA in the step (1) in a single tube in the presence of a group of primers and probes; (3) determining the presence or absence of group B streptococci in the sample, and if not, determining the presence or absence of other Streptococcus bacteria, the set of primers and probes including a first primer pair and a first probe for detecting a conserved region in the Streptococcus genome; a second primer pair and a second probe for detecting a first conserved region in the group B streptococcus genome; a third primer pair and a third probe for detecting a second conserved region in the group B Streptococcus genome that is different from the first conserved region.

Further, the set of primers and probes further comprises a fourth primer pair and a fourth probe for detecting an internal standard gene.

Further, the conserved region of the Streptococcus genome is from a 16S rRNA gene, a 23S rRNA gene, or a 16S-23S rDNA spacer.

Further, the first conserved region is from a Sip or Cfb gene.

Further, the second conserved region is from a Sip or Cfb gene.

Further, the base sequences of the first primer pair and the first probe are respectively SEQ ID NO. 1-3.

Further, the base sequences of the second primer pair and the second probe are respectively SEQ ID NO. 4-6.

Further, the base sequences of the third primer pair and the third probe are respectively SEQ ID NO. 7-9.

Further, the base sequences of the fourth primer pair and the fourth probe are respectively SEQ ID NO 10-12.

Further, the fluorescence reporter gene and the quenching group of each probe are selected from any one of FAM-TARMA/BHQ1, VIC/HEX/JOE-TAMRA/BHQ1, ROX/Texas Red-BHQ2 and Cy5/LC Red640-BHQ2/BHQ 3.

The invention also provides a kit for detecting streptococcus, which comprises a fluorescent PCR reaction solution, wherein the fluorescent PCR reaction solution comprises oligonucleotides, and the oligonucleotides comprise (1) a first primer pair and a first probe for detecting a conservative region in a streptococcus genome; (2) a second primer pair and a second probe for detecting a first conserved region in the group B streptococcus genome; (3) a third primer pair and a third probe for detecting a second conserved region in the group B Streptococcus genome that is different from the first conserved region.

Further, the oligonucleotide comprises (4) a fourth primer pair and a fourth probe for detecting an internal standard gene.

Further, the conserved region of the Streptococcus genome is from a 16S rRNA gene, a 23S rRNA gene, or a 16S-23S rDNA spacer.

Further, the first conserved region is from a Sip or Cfb gene.

Further, the second conserved region is from a Sip or Cfb gene.

Further, the base sequences of the first primer pair and the first probe are respectively SEQ ID NO. 1-3.

Further, the base sequences of the second primer pair and the second probe are respectively SEQ ID NO. 4-6.

Further, the base sequences of the third primer pair and the third probe are respectively SEQ ID NO. 7-9.

Further, the base sequences of the fourth primer pair and the fourth probe are respectively SEQ ID NO 10-12.

Further, the fluorescence reporter gene and the quenching group of each probe are selected from any one of FAM-TARMA/BHQ1, VIC/HEX/JOE-TAMRA/BHQ1, ROX/Texas Red-BHQ2 and Cy5/LC Red640-BHQ2/BHQ 3.

Further, the kit also comprises a nucleic acid extracting solution 1 and a nucleic acid extracting solution 2, and is characterized in that the nucleic acid extracting solution 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1-2% SDS, and the nucleic acid extracting solution 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100

The invention also provides a kit for detecting streptococcus, which comprises PCR reaction liquid, enzyme mixed liquid, positive control, negative control and nucleic acid extracting solution, and comprises (1) primers and probes for detecting streptococcus, wherein the base sequences of the primers and the probes are respectively SEQ ID NO. 1-3; (2) primers and probes for detecting group B streptococcus Sip genes, wherein the base sequences of the primers and the probes are respectively SEQ ID NO. 4-6; (3) the primers and the probes for detecting the group B streptococcus Sip gene have base sequences of SEQ ID NO of 7-9: (4) the primers and the probes for detecting the reference gene have base sequences of SEQ ID NO 10-12 respectively.

Further, the concentration of each primer and probe is in the range of 75-300 nM.

Further, the nucleic acid extracting solution comprises a nucleic acid extracting solution 1 and a nucleic acid extracting solution 2, wherein the nucleic acid extracting solution 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1-2% SDS; the nucleic acid extract 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100.

Further, the PCR reaction solution further comprises 10 to 50mM Tris (pH 8.0 to 9.2), 10 to 50mM KCl, 10 to 20mM ammonium sulfate, 0.01 to 0.1% Tween20, 0.2 to 2mg/mL BSA, 0.1 to 0.3mM dATP, 0.1 to 0.3mM dTTP, 0.1 to 0.3mM dCTP, 0.1 to 0.3mM dGTP, 0.1 to 0.3mM dUTP, and 3 to 6mM magnesium chloride.

Further, the kit comprises the following operation procedures: (1) extracting sample DNA by a two-step method: firstly, 1mL of sample is taken and centrifuged at 12000rpm, the supernatant is discarded, 0.5mL of nucleic acid extracting solution 1 is added, the mixture is evenly mixed in a vortex mode, and the supernatant is discarded; secondly, adding 100 mu L of nucleic acid extracting solution 2, boiling for 10min at 100 ℃, centrifuging at 12000rpm, and reserving supernatant nucleic acid for later use; (2) preparing a PCR reagent: mixing the PCR reaction solution and the enzyme mixed solution uniformly, and respectively adding the sample DNA extracted in the step (1) and negative quality control and positive quality control; (3) and (3) fluorescent PCR detection: performing fluorescence PCR amplification, and determining whether streptococcus and member B group streptococcus exist in the sample after the amplification is finished.

Has the advantages that: (1) the invention selects two different conserved regions (for example, one conserved region is from an Sip gene, and the other conserved region is from a Cfb gene) in the genome of the group B streptococcus to design two groups of primers and probes, simultaneously designs one group of primers and probes as an internal control according to a conserved sequence (for example, a conserved sequence from a 16S rRNA gene, a 23S rRNA gene or a 16S-23S rDNA spacer region) in the genome of the streptococcus, and introduces a human genome housekeeping gene beta-globin as an internal reference to establish a dual-quality control and dual-target verification system, thereby effectively avoiding the result abnormality caused by a series of factors such as clinical sample sampling errors, nucleic acid extraction errors and the like, and more importantly, solving the problem of the omission of the group B streptococcus; (2) since group B streptococci are also a member of the genus Streptococcus, when group B streptococci exist in a clinical sample, under the condition that the conserved regions of the Sip and Cfb genes to be detected are not mutated, obvious S curves appear when 16S rRNA, Sip and Cfb genes are subjected to fluorescence PCR detection, so that a plurality of indexes are provided for judging whether group B streptococci exist in the clinical sample, and the occurrence of false positives can be further reduced; (3) since the group B streptococcus is subjected to different evolutionary stresses in different human environments and has different mutation rates, when the group B streptococcus exists in clinical samples, the group B streptococcus is not detected due to mutation of genes to be detected (Sip is taken as an example), whether the group B streptococcus exists can be judged according to whether obvious S curves appear in fluorescent PCR detection aiming at 16S rRNA and Cfb genes, and therefore group B streptococcus omission caused by gene sequence mutation is reduced as much as possible; (4) when group B streptococcus is not present in the clinical sample, no obvious S curve appears in the fluorescent PCR detection aiming at Sip and Cfb genes, in this case, if the fluorescent PCR detection aiming at 16S rRNA gene is carried out, the S curve does not appear, which indicates that the streptococcus is not present in the clinical sample, and if the fluorescent PCR detection aiming at 16S rRNA gene is carried out, the S curve appears, which indicates that streptococcus except the group B streptococcus is present in the clinical sample, which is helpful for a clinician to carry out further pathogen investigation; (5) the kit can detect the group B streptococcus from a complex sample, has the advantages of high sensitivity, good specificity, quick and objective detection result and the like, and provides a reliable result for diagnosing the infection of the streptococcus and the group B streptococcus which are members of the streptococcus.

Drawings

FIG. 1: the invention aims at the detection result of the nucleic acid extracted by the alkaline lysis method.

FIG. 2: results of detection of nucleic acids extracted by the magnetic bead method as a control.

FIG. 3: the invention detects the result of the positive reference product P1.

FIG. 4: the invention detects the result of the positive reference product P2.

FIG. 5: the invention detects the result of the positive reference product P3.

FIG. 6: the invention detects the result of the positive reference product P4.

FIG. 7: the invention detects the result of the positive reference product P5.

FIG. 8: the invention aims at the detection result of the negative reference product N1-N10.

FIG. 9: the invention discloses a Cy5 channel Cfb gene detection sensitivity preliminary judgment result graph.

FIG. 10: the invention relates to a result chart of the initial judgment of the detection sensitivity of the Sip gene of the ROX channel.

FIG. 11: the present invention is directed to a graph showing the results of verifying the detection sensitivity of Cy5 channel Cfb gene at 500 copies/mL.

FIG. 12: at 500copies/mL, the invention aims at the verification result chart of the detection sensitivity of the Sip gene of the ROX channel.

FIG. 13: the present invention is directed to a graph showing the results of verifying the detection sensitivity of Cy5 channel Cfb gene at 1000 copies/mL.

FIG. 14: the invention aims at a verification result chart of the detection sensitivity of the Sip gene of the ROX channel when the copy/mL is 1000 copies/mL.

FIG. 15: 2000copies/mL, the present invention is directed to a graph showing the results of verifying the detection sensitivity of Cy5 channel Cfb gene.

FIG. 16: 2000copies/mL, the invention aims at the verification result chart of the ROX channel Sip gene detection sensitivity.

Detailed Description

In the method for detecting group B streptococcus, primers and probes are designed by using a conserved sequence in group B streptococcus, and then a housekeeping gene in human genome DNA is detected to detect whether group B streptococcus exists in a clinical sample. However, group B streptococci are subject to different evolutionary stresses in different human environments, with different mutation rates. In order to reduce the omission of group B streptococcus caused by gene sequence mutation as much as possible, the invention selects two sections of conserved sequences in a group B streptococcus genome to design two groups of primers and probes, simultaneously designs one group of primers and probes as an internal control according to one section of conserved sequences in the streptococcus genome (for example, the conserved sequence is from a 16S rRNA gene), and introduces a human genome housekeeping gene (for example, beta-globin) as an internal reference to establish a dual-quality control and dual-target verification system, thereby effectively avoiding result abnormality caused by a series of factors such as clinical sample sampling errors, nucleic acid extraction errors and the like, and more importantly, solving the problem of the omission of the group B streptococcus. Based on this, primers and probes designed are shown in Table 1.

TABLE 1 primers and probes

Table 1 contains 4 total TaqMan probes, each of which has a fluorescent reporter group at the 5 'end (x1, x2, x3 and x4) and a quencher group which emits fluorescence or does not emit fluorescence at the 3' end (y1, y2, y3 and y 4). The fluorescent reporter groups of the 4 probes can be selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640, LC RED705 and the like; the quenching group can be selected from BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, NFQ and the like, and is selected according to the principle that the fluorescence absorption spectrum of the quenching group overlaps with the emission spectrum of the fluorescence reporter group. The preferred combination of fluorescent reporter and quencher groups for these four probes in Table 1 is any of FAM-TARMA/BHQ1, VIC/HEX/JOE-TAMRA/BHQ1, ROX/Texas Red-BHQ2 and Cy5/LC Red640-BHQ2/BHQ 3. Preferably, the fluorescent reporter group is different for each probe, so that four-channel detection can be performed simultaneously in a single tube.

Preferred fluorescent reporters are FAM, ROX and Cy5 because these three fluorescent reporters have the advantages of low background fluorescence and high fluorescence detection signal in the fluorescent quantitative PCR detection.

Before performing fluorescent PCR, nucleic acid (DNA) in a clinical sample needs to be extracted, and various methods such as an alkaline lysis method, a magnetic bead method and a column extraction method are used for extracting the nucleic acid. For convenience of illustration, the invention adopts an alkaline lysis method to extract nucleic acid in a clinical sample, and the adopted nucleic acid extracting solution comprises a nucleic acid extracting solution 1 and a nucleic acid extracting solution 2, wherein the nucleic acid extracting solution 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1-2% SDS; the nucleic acid extract 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100. However, it should be noted that although the method of alkaline lysis is used to extract nucleic acid from a clinical sample, the present invention is not limited to this nucleic acid extraction method, and magnetic bead method and column extraction method can be used to extract nucleic acid from a clinical sample.

For convenience of explanation, in the following examples, FAM-BHQ1 was used as a fluorescent reporter and a quencher for a probe for detecting the Streptococcus 16S rRNA gene, ROX-BHQ2 was used as a fluorescent reporter and a quencher for a probe for detecting the group B streptococcal Sip gene, CY5-BHQ3 was used as a fluorescent reporter and a quencher for a probe for detecting the group B streptococcal Cfb gene, and VIC-BHQ1 was used as a fluorescent reporter and a quencher for a probe for human genome housekeeping gene β -globin. The strains used in the examples of the present invention were purchased from ATCC and used for the preparation of positive quality control and reference in the kit.

The following examples further illustrate the invention. These examples are not intended to limit the scope of the invention but rather to provide a further understanding of the invention.

Example 1: extraction of nucleic acids from clinical samples

For convenience of explanation, the vaginal secretion of pregnant and lying-in women is taken as a clinical sample in the present embodiment.

Taking 1mL of vaginal secretion sample of a pregnant and lying-in woman, centrifuging the sample in a 1.5mL centrifuge tube at 12000rpm for 10min, sucking and discarding supernatant, adding 500 mu L of nucleic acid extracting solution 1, shaking and uniformly mixing, centrifuging the mixture at 1200rpm for 10min, and sucking and discarding supernatant; adding 50 μ L of nucleic acid extract 2, shaking, mixing, and water bath or dry bath at 100 deg.C for 10min to obtain supernatant as DNA extract. Wherein the nucleic acid extract 1 comprises 50mM Tris, 20mM NaCl and 1% SDS; nucleic acid extract 2 contained 50mM Tris, 20mM NaCl, 0.5% mercaptoethanol, and 2% Chelex-100.

The nucleic acid extraction method is an alkaline cracking method, and a two-step method is adopted to respectively wash and extract nucleic acid from clinical samples. Nucleic Acid (DNA) extraction kit (magnetic bead method) produced by ikang biotechnology (hang zhou) ltd (No. 20150133 Zhe hang machinery) is used as a control to extract nucleic acid from the same clinical sample, the kit of the present invention is used to detect human genome DNA housekeeping gene β -globin, and the detected Ct values are compared, and the results are shown in fig. 1 and fig. 2. As can be seen from FIGS. 1 and 2, the nucleic acid extraction method of the present invention has substantially the same detection results as the magnetic bead extraction method, and also has the characteristics of simple operation, low reagent cost, etc.

Example 2: detection of group B streptococci using fluorescent PCR method

In the embodiment, the kit is used for carrying out fluorescent quantitative PCR detection on the group B streptococcus, and comprises PCR reaction liquid, enzyme mixed liquid, positive quality control and negative quality control. Preferably, the kit may further comprise the nucleic acid extracting solution 1 and the nucleic acid extracting solution 2 in example 1. The PCR reaction solution preparation system is shown in Table 2. The enzyme mixture formulation is shown in Table 3. The positive quality control is a group B streptococcus culture, and the negative quality control is 0.9% NaCl solution.

TABLE 2 PCR reaction solution preparation System TABLE (1 person)

TABLE 3 enzyme mixture preparation system (1 part)

When detecting pathogen group B streptococcus in clinical samples, the PCR reaction solution and the enzyme mixed solution are mixed uniformly, and the required number of samples is calculated by n parts [ n is the number of clinical samples +1 tube positive control +1 tube negative control ]. mu.L of the PCR reaction solution was added to different reaction tubes, and then 4. mu.L of the DNA solution extracted in example 1, 4. mu.L of the negative control, and 4. mu.L of the positive control were added to the different reaction tubes, respectively.

The reaction tubes were placed in a fluorescent PCR apparatus (ABI 7500) in a certain order, and PCR amplification was carried out according to the following procedure, as shown in Table 4:

TABLE 4 fluorescent PCR amplification procedure

After the PCR amplification is finished, whether group B streptococcus exists is detected according to Ct values of a FAM channel, a VIC channel, a ROX channel and a Cy5 channel, and the detection results are judged as shown in Table 5.

TABLE 5 test results Cutoff values and results interpretation

The common test results of clinical samples are shown in table 6, and other abnormal results need to be interpreted in combination with clinical tests.

TABLE 6 interpretation of common results in clinical samples

Example 3: internal clinical sample validation

Clinical samples were collected from the Hangzhou Edikang medical testing center for a total of 300 pregnant and lying-in woman vaginal secretion samples for internal clinical evaluation.

300 samples of cervical secretions from pregnant and lying-in women were isolated as described in example 1 and subjected to fluorescent PCR amplification as described in example 2, and the results were recorded as test group 1.

According to the method described in example 2, Cfb genes were detected individually by using sequences SEQ ID NO: 7-9 (experiment group 2), Sip genes were detected individually by using sequences SEQ ID NO: 4-6 (experiment group 3), PCR reaction solution preparation systems for group B streptococci were prepared in cooperation with internal standard gene detection, and fluorescence PCR amplification was performed on nucleic acids in 300 of the same maternal cervical secretion samples in parallel, and the detection results were recorded. The analysis for the three sets of test results is shown in table 7.

Table 7: double-target system and single-target detection result consistency analysis

The results of the above results were not the same as those of experiment group 1 in 1 sample for both experiment group 2 and experiment group 3. Sanger sequencing verification is carried out on samples with inconsistent results, and the verification result is consistent with the detection result of the experimental group 1, which shows that the double-target detection system adopted by the invention can effectively avoid the missed detection of the positive specimen, thereby improving the detection accuracy.

In order to further verify the accuracy of the dual-target detection system, a group B streptococcus nucleic acid detection reagent (fluorescence PCR method) (a primer probe designed according to Cfb gene, an upstream primer: 5'-AGCAATCACACATGCTGTTGGA-3', a downstream primer: 5'-TAATGCTGTTTGAAGTGCTGCT-3', a probe: 5 'FAM-CAGTTGAATCCAAATGTTACGGTACAAC-TAMRA 3', a contrast reagent 1 below) and a group B streptococcus nucleic acid determination reagent (fluorescence PCR method) (a primer probe designed according to Sip gene, a contrast reagent 2 below, an upstream primer: 5'-TTGACATCGACAATGGCAGC-3', a downstream primer: 5'-TAACACTTGCCACTCTAGGG-3', a probe: 5 'FAM-AACAGATACGACGTGGACAG-TAMRA 3', a contrast reagent 2 below) adopted in the prior art are respectively used as contrast reagents, and the nucleic acid in 300 identical cervical secretion samples of pregnant and lying-in women is tested in parallel. The results are shown in Table 8.

TABLE 8 test results of clinical specimens

The results in Table 8 show that the results of 2 samples of control 1 are not the same as those of the present invention; the results for 1 sample of control reagent 2 were not the same as those in the present invention. Sanger sequencing verification is carried out on samples with inconsistent results, and the verification result is consistent with the detection result of the experimental group 1, which shows that the double-target detection system adopted by the invention can effectively avoid the missed detection of the positive specimen, thereby improving the detection accuracy.

Example 4 reference System establishment and evaluation

In this example, the following reference materials were set for the kit of the present invention to evaluate the performance. Setting a positive reference product: group B streptococci (serotypes Ia, Ib, II, III and V) were selected at a concentration of approximately 5X 10⁵The standard strain culture of copies/mL was used as a positive reference for the kit (P1-P5). Negative reference setting: 1 part of chlamydia trachomatis sample, 1 part of neisseria gonorrhoeae positive sample, 1 part of ureaplasma urealyticum positive sample, 1 part of HSV-2 positive sample, 4 parts of HPV positive sample (including 1 part of each of HPV16, 18, 52 and 58), 1 part of human genome DNA and 1 part of TE buffer solution are selected as negative reference substances (sequentially marked as N1-N10).

The reference substance set in the kit was evaluated for the rate of coincidence of yin and yang according to the method described in example 2. The results are shown below:

the detection results of the positive reference products P1-P5 are that the detection results of FAM, ROX and Cy5 channels are positive, and the detection result of the VIC channel is negative. As shown in fig. 3-7.

The detection results of 9 negative reference products, namely FAM, ROX and Cy5 channels of the negative reference products N1-N9 are negative, and the detection result of only the VIC channel is positive; the detection result of the negative reference product N10 is that the FAM, VIC, ROX and Cy5 channel results are all negative. As shown in fig. 8.

Example 5 assay sensitivity

In this example, the products of the high concentration of standard strain cultures (about 10) were individually cultured against the most common serotype III of group B Streptococcus⁵copies/mL) and the gradient diluted sample is detected according to the method in the embodiment 2, the lower limit of the detection of the kit is preliminarily judged, and the preliminary judgment is about 1000 copies/mL. As shown in FIGS. 9 to 10.

The test was repeated 20 times according to the kit described in example 2 for different concentrations (500, 1000, 2000copies/mL, respectively) of the most common serotype III standard strain culture of group B Streptococcus at high concentrations, and the lower detection limit of the kit was confirmed at a detection rate of 95%. The detection results are shown in tables 8 and 9 in detail, wherein the table 8 is the ROX channel detection result statistics, the table 9 is the Cy5 channel detection result statistics, the detection rates of the Sip gene amplification primer probes to 500, 1000 and 2000copies/mL standard bacteria are 80%, 100% and 100% respectively, and the detection rates of the Cfb gene amplification primer probes to 500, 1000 and 2000copies/mL standard bacteria are 80%, 95% and 100% respectively; in summary, the lower detection limit of the kit is 1000 copies/mL. As shown in FIGS. 11-16.

TABLE 9 statistics of the lower limit results of the detection of Sip genes in ROX channels

TABLE 10 statistics of Cy5 channel Cfb Gene detection lower limit results

Example 6 assay precision analysis

The kit of example 2 was used to test 1 sample with positive concentration (greater than 10)⁵The copies/mL), 1 weak positive sample (about 2000copies/mL) and 1 negative sample are respectively tested for 8 times, the test results of different channels are counted, the details are shown in Table 10, and the CV of the Ct value detected by the kit is less than 5% according to the results, so that the kit disclosed by the invention is good in precision in batch.

TABLE 11 precision sample test results

Sequence listing

<110> Liduo (hong Kong) Co., Ltd

<120> method, oligonucleotide and kit for detecting streptococcus

<150> 201911146519.X

<151> 2019-11-21

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tcttaaagcc agtctcagtt cggat 25

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gacttcgggt gttacaaact ctcgt 25

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgtaggctgc aactcgccta catgaagtc 29

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgttgcagac caaaaagttt ctctc 25

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cttgtgctaa tacttctttt gatttcaaa 29

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctggcgcaga agaatatgtc ttcattggc 29

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aacttgttgt accgtaacat ttggat 26

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtacttaac gtcaaagaat ttaaagt 27

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caactgaact ccaacagcat gtgtgattgc 30

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agacaatggt gcatctgact cctg 24

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccaccaactt catccacgtt cac 23

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tggagaagtc tgccgttact gccctgt 27

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agcaatcaca catgctgttg ga 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

taatgctgtt tgaagtgctg ct 22

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cagttgaatc caaatgttac ggtacaac 28

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttgacatcga caatggcagc 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

taacacttgc cactctaggg 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aacagatacg acgtggacag 20