RU2664454C2 - Method for genetic diagnosis of mutant allele that causes short spine syndrome or brachyspine syndrome in cattle and a test system for implementation thereof - Google Patents

Abstract

FIELD: veterinary science.SUBSTANCE: invention relates to veterinary medicine and is intended for diagnosis of mutant allele that causes short spine syndrome or brachyspine syndrome in cattle. DNA is isolated from biological material, polymerase chain reaction is set in real time (PCR-RT) using a reaction mixture with BY. Reaction results are further evaluated: BY is not detected, if a value is positive on channel Green and if a value is negative for channel Yellow, BY is detected if a value is positive on channels Green and Yellow.EFFECT: invention provides a shorter PCR-RT time and ability to monitor the validity of the study.1 cl, 3 dwg, 2 tbl, 1 ex

Description

The invention relates to the field of molecular genetics and can be used in veterinary medicine and animal husbandry to diagnose a mutation that causes a hereditary disease: a short spine or brahispin (Bovine Brachyspina Syndrome, BYS) in cattle. The synthesized sequence set of primers and allele-specific probes allows the identification of the BYS mutation in the FANCI 21 chromosome gene using real-time PCR (PCR-RV). The mutation is associated with a deletion, i.e. with a loss of 25-27 exons out of 37 in the 3.3 kb region of the FANCI gene Holstein cattle. Congenital disease, not related to gender, is inherited in an autosomal recessive manner. In the medical literature, this disease is called Fanconi anemia (FANCI). The disease is named after the Swiss pediatrician Guido Fanconi, who was the first to find it.

Currently, about 60 of 400 known hereditary diseases that are detected at the DNA level are described in cattle (ICAR Guidelines, 2011; Marzanova S.N. et al., 2015). They cause serious morphological and functional abnormalities, negatively affect the health and productivity of the animal. Therefore, an important task of veterinarians and livestock specialists is the timely diagnosis and eradication of the source that causes this disease. Knowledge of the molecular structure, the definition of a heterozygous animal carrier, is the basis for the effective fight against hereditary disease.

The speed and accuracy of the diagnosis of brahispin was significantly improved due to the establishment of the genetic nature of this molecular disease (Agerholm et al., 2006; 2007; 2010; Thomsen B. et al., 2006; Testoni S. et al., 2008; Buck BC et al., 2010; VanRaden PM, et al., 2011; Charlier C. et al., 2012).

In a number of European countries (Agerholm JS et al., 2006; Testoni S. et al., 2008; Buck BC et al., 2010), the mutation was traced to the elite American Holstein bull Sweet Haven Tradition (HOUSAM 1682485) . Although, in the Canadian case, a more distant ancestor is supposed (Agerholm J.S. et al., 2006). The descendants of Sweet Haven Tradishin were widely used in breeding programs of dairy cattle in many countries of the world, and the BY mutant allele spread throughout the world.

Brahispin Syndrome (Bovine Brachyspina Syndrome, BYS) is a genetic defect in the Holstein cattle, which leads either to early termination of pregnancy. Abortion, the most common symptom, or calving with stillborn calves, which is a rarer case when an individual is homozygous for a recessive lethal allele. The defect was first identified in Denmark in 2006 (Agerholm J.S. et al., 2006). Later, similar cases were reported in the Netherlands (Agerholm JS, Peperkamp K., 2007), Italy (Testoni S. et al., 2008), Germany (Buck BC et al., 2010), Canada (Agerholm JS et al., 2010), China (Lingzhao Fang et al., 2013) and others. Stillborn calves were characterized by a significant decrease in body weight, despite a normal or slightly prolonged period of pregnancy, a noticeable shortening of the spine, long and thin limbs, and the presence of a lower bite - brachygnathism , a clear violation in the development of internal organs, for example, dysplasia of the kidneys and genital glands. At the same time, the defect may be the reason for some violations of the course of pregnancy, due to frequent cases of abortion and long intervals between calving (Charlier C. et al., 2012). Most cases of abortion are naturally accompanied by fetal death during embryonic or early fetal development. In this case, according to the signs of brahispin, it can be similar to another hereditary disease - a complex of spinal anomalies (CVM) (Thomsen B. et al., 2006), causing a decrease in herd fertility and unpredictable economic losses for animal owners.

Earlier studies have established a gene that causes BYS in positions encompassing a 3.3 kb deletion. in the FANCI gene 21 cattle chromosomes and is diagnosed as single nucleotide polymorphism (SNP). In 2010, a group of scientists from different European countries created the international patent WO / 2010/012690 for the diagnosis of brahispina (Georges M., Coppieters W., Charlier C., Agerholm JS, Fredholm M. 2010. A genetic test for Brachyspina and fertility in cattle Patent application WO / 2010/012690).

In 2012, the cause of the mutation causing brahispina (Bovine Brachyspina Syndrome, BYS) was successfully identified by sequencing. It turned out to be a 3.3 kb portion of the FANCI DNA gene to be deleted, i.e. loss of 25-27 exons out of 37 in Holstein cattle responsible for Fanconi syndrome or anemia. Deletion led not only to the removal of part of the DNA, but also the replacement of some amino acids by others in the synthesized proteins. The usual carrier frequency is less than 1 / 100,000, but its occurrence in the Holstein breed is 7.4% or higher. Such a sharp discrepancy between the two figures is possibly associated with the death of homozygous calves in the early stages of pregnancy (Charlier C. et al., 2012).

In the Russian Federation and abroad, there are no analogues to this development. At present, outstanding manufacturers and repair young stock of breeding enterprises of the Russian Federation are being tested according to the developed method for carriage of the mutant BY allele. The results without fail, along with blood groups, kappa-casein, CVM and BLAD are regularly published in individual cards of breeding bulls.

A genetic test is known in the art for identifying carriers of the recessive brahispina allele in cattle. This test allows you to determine whether a bull is a BY carrier by analyzing its genomic DNA or RNA. The method can be used to perform selection using markers or a genomic kit to increase fertility (Patent for the invention WO / 2010/012690 publ. 04.02.2010).

This invention is accepted as the closest analogue. A distinctive feature of the claimed invention from the identified analogue is the ability to determine BY by the method of real-time PCR (PCR-RV), using primers and allele-specific probes. The BY mutation point is the same as in the indicated analogue, however, the detection approach is fundamentally different.

In the claimed invention is the identification of polymorphism at the FANCI locus using PCR-RV and using TaqMan probes. The study time of this polymorphism takes no more than 2 hours.

The method and test system proposed for patenting for its implementation in comparison with all the analogues studied has the following advantages: specific structure of primers and probes, the possibility of real-time polymerase chain reaction (PCR-RV), which ensures the speed of diagnostics and obtaining the result in 2 hours.

The technical result of the claimed invention is the possibility of diagnosing brahispin or short spine (Bovine Brachyspina Syndrome) in cattle by real-time PCR, reducing the time to complete the polymerase chain reaction in real time (PCR-RV) (after 2 hours the result is obtained); not laborious; the ability to perform repetitions; to control the accuracy of the study.

The claimed technical result is achieved due to the fact that DNA is extracted from biological material, the polymerase chain reaction is set up in real time using a reaction mixture with BY, a cycling process in which denaturation, annealing of primers and synthesis of a new DNA chain occurs at a temperature of 64 ° C (at the first cycle of 10 repetitions, at the second cycle of 30 repetitions). Then, the reaction results are evaluated: BY was not detected with a positive value on the Green channel and a negative value on the Yellow channel, BY was detected with a positive value on the Green and Yellow channels.

The test system for the diagnosis of a mutant allele that causes a short spine or brahispin in cattle includes a PCR mixture for the reaction to detect the BY allele, a diluent, thermostable Taq polymerase enzyme, positive for the detection of normal and mutant BY alleles, and negative controls.

To carry out gene diagnostics of the BY allele in cattle using real-time PCR (PCR-RV), primers and allele-specific probes were used, having the following sequences:

The present invention provides a set of primers and probes for diagnosing a mutant BY allele in a black-motley genealogical root, which includes such well-known breeds as Holstein, black-motley, Kholmogorsk, Yaroslavl, Tagil, in addition, red-motley breed, as well as synthetic populations with an admixture of Holstein blood.

The optimal composition of the reaction mixture for real-time polymerase chain reaction is claimed, a combination of primers, the necessary and sufficient ratio of the components of the reaction mixture are given, and the PCR formulation mode is determined, which is important when conducting PCR analysis. The system is designed to carry out 100 reactions with a volume of 25 μl (20 μl + 5 μl of the test sample).

Primers and allele-specific probes were selected using the Oligo 6.0 application package based on the analysis of the nucleotide sequences of the FANCI DNA locus published in the NSBI database.

An example of a polymerase chain reaction using the claimed kit.

The source for the diagnosis is pre-isolated DNA from samples of biological material (whole blood, sperm, embryos, milk, wool or skin).

The reaction mixture for PCR analysis has the following composition: 50 mm KCl, 50 mm TRIS-HCl, 250 nm dNTP, 2.5 mm MgCl ₂ , 2.5 units DNA polymerases, primers at a concentration of 200 nM, fluorescent probes at a concentration of 100 nM, having specific sequences:

Preparation for the reaction was carried out as follows.

The required volume of components was calculated based on the number of test samples plus 4 (three positive control samples and one negative control sample). A set of components for the reaction are presented in table No. 1.

The components were thawed, mixed and centrifuged. A mixture of components was prepared by adding them in the order shown in table 2, mixed and centrifuged.

Reaction Profile:

1. Holding the temperature at 94 ° C for 3 minutes, one repeat.

2. Cycle 1

94 ° C - 20 s

61 ° C - 20 s

64 ° C - 30 s

Repeat the cycle 10 times.

3. Cycle 2 (with detection)

94 ° C - 20 s

61 ° C - 20 s

64 ° С - 30 s - detection through channels: Green, Yellow

Repeat the cycle 30 times. In the process of cycling, denaturation, annealing of the primers and synthesis of a new DNA chain occurs at a temperature of 64 ° C. During the first cycling, 10 repetitions were carried out - no detection, since the data are not representative. During the second cycle, 30 repetitions were carried out - as a result, stable data were obtained, which are final in terms of diagnosis.

The creation of the PCR-RV template, as well as the analysis of the results of the study, was performed using the Rotor-Gene 6000 1.8 software to the Rotor Gene Q instrument (Corbett Research, Australia).

The result of the analysis is the determination of a mutant allele that causes brahispin (BYS) in cattle.

In the claimed invention used the technology of allele-specific TaqMan probes. In this case, the fluorescent probe is an oligonucleotide complementary to the PCR product.

The probe is labeled with a fluorophore and a fluorescence quencher, and both terminal and internal labeling of the oligonucleotide can be used. In the absence of a target, i.e. the sequences complementary to the probe, the fluorophore and the quencher come closer, as a result, the fluorescence of the probe is suppressed. The quenching process is based on the mechanism of fluorescence resonance energy transfer. In the presence of a DNA fragment complementary to the probe, the probe hybridizes with the amplicon, which leads to its cleavage during synthesis due to the 5'-exonuclease activity of Taq polymerase. The signal intensity increases on the fluorescence channel corresponding to the fluorophore with each PCR cycle, in proportion to the accumulation of amplicons.

Allelespecific probes are effective in detecting BYS mutations in the studied fragment of the genome. For a locus with a mutation, a pair of probes is selected, so that one of them specifically hybridizes at the temperature of the chain synthesis with the normal (wild) type allele, and the second probe with the mutant type allele.

These probes are labeled with different fluorophores, and the results of reactions with them are recorded on two different detection channels of the PCR-RV device. To determine the BT and BY alleles, 2 channels are used - Green and Yellow. The normal BT allele gives rise to a signal on the Green channel, detected by the FAM fluorophore, and the mutant BY allele, on the Yellow channel, detected by the R6G fluorophore.

The results are interpreted based on the presence or absence of the intersection of the fluorescence curve with a threshold line set at an appropriate level.

A threshold transition is a characteristic of the number of copies of the DNA of an individual gene. With the optimal number of DNA copies, the curve is located between two threshold values of 10 and 15 amplification cycles.

When moving the curve to the left along the abscissa, this indicates a high concentration of DNA, if to the right, about its lack of reaction.

All samples whose fluorescence plots cross the threshold line will be considered positive for a specific channel, i.e. containing an allele corresponding to the fluorescence channel.

The resulting picture of the reaction results using these probes on control samples is given below when determining the mutant BY allele.

In fig. 1 results of studies of a homozygous wild-type or healthy animal or non-carrier, fluorescence is represented by two lines.

The curve without markers is located between 10 and 15 cycles above the red threshold line. The second curve with circles below the threshold line. This situation is characteristic of a healthy animal under investigation.

In fig. 2, the results of a study of a heterozygous animal or carrier of a mutant BY allele are presented. As seen in fig. 2, both fluorescence curves cross the threshold line set at the appropriate level in the designated area, between 10 and 15 PCR-RV cycles.

At the same time, it should be noted that the curve with circles is located above the curve without markers. This picture is characteristic of a heterozygous animal or carrier of a mutation.

When developing this method, it was not possible to obtain sick calves empirically in the household. In this regard, for the first time it was possible to model synthetic recessive genotypes of sick calves using the FANCI gene, in laboratory conditions, based on knowledge of the structures of the mutant BY allele.

In fig. Figure 3 shows a model sample of an animal homozygous for the mutant BY allele. As seen in fig. 3, the model sample reflects the situation characteristic of a sick calf. In this case, the curve with circles is located above the threshold, the fluorescence curve without circles, characteristic of a healthy animal, is below the threshold line.

The prognosis of the resulting offspring depending on the presence or absence of a mutant BY allele. The presence of the BY allele is a striking example of carriage of a mutation on the one hand, and on the other, manifestations of single nucleotide polymorphism or SNP (single nucleotide polymorphism) in DNA in cattle.

The main advantages of the claimed invention are:

1. The ability to certify elite animals of a black-and-white genealogical root and red-and-white breed according to the BYS mutation using the created Set of presented primer and probe sequences will allow the directional formation of the allele pool of bulls, bull-reproducing groups of cows, repair young stock, and also accumulate breeding material (bank seed and embryos) from healthy animals.

2. The presence of a genetic passport for the BYS mutation of elite animals will allow for efficient breeding and breeding work in breeding herds of black-motley genealogical root and red-motley breed.

3. When importing breeding material into the Russian Federation from abroad, a genetic passport is required.

4. In order to stop the hereditary disease, purchased animals after quarantine must be re-tested for the presence of a mutant BY allele.

Literature

1. Marzanova S.N., Nagorny V.A., Devrishov D.A., Alekseev Y.I., Konovalova N.V., Tokhov M.Kh., Yeskin G.V., Turbina I.S., Lukashina A.A., Marzanov N.S. The founder effect and genogeography of CV- and BL-mutations in black-and-white cattle // Doklady RAAS. 2015. No.6. S. 44-47.

2. Agerholm J.S., DeLay J., Hicks B., Fredholm M. First confirmed case of the bovine brachyspina syndrome in Canada // Can. Vet. J. 2010. Vol. 51. P. 1349-1350.

3. Agerholm J.S., McEvoy F., Arnbjerg J. Brachyspina syndrome in a Holstein calf // J. Vet. Diagn. Invest. 2006. Vol. 18. P. 418-422.

4. Agerholm J.S., Peperkamp K. Familian occurrence of Danish and Dutch cases of the bovine brachyspina syndrome // BMC Vet. Res. 2007. Vol. 3.P. 8.

., Kuiper H., Baumgartner W., Distl O. Missbildungen an der Wirbelsaule mit multiplen Organanomalien bei einem Kalb der Rasse Deutsche Holsteins [Vertebral and multiple organ malformations in a black and white German Holstein calf]. Berl Munch Tierarztl Wochenschr. 2010. Vol. 123. P. 251-255. In German. Abstract in English.5. Buck BC, Ulrich R.,

., Kuiper H., Baumgartner W., Distl O. Missbildungen an der Wirbelsaule mit multiplen Organanomalien bei einem Kalb der Rasse Deutsche Holsteins [Vertebral and multiple organ malformations in a black and white German Holstein calf]. Berl Munch Tierarztl Wochenschr. 2010. Vol. 123. P. 251-255. In German. Abstract in English.

6. Charlier C., Agerholm JS, Coppieters W., Karlskov-Mortensen P., Li W., de Jong G., Fasquelle C., Karim L., Cirera S., Cambisano N., Ahariz N., Mullaart E ., Georges M., Fredholm M. A deletion in bovine FANCI gene compromises fertility by causing fetal death and brachyspina // PLoS One. 2012. Vol. 7 (8): e43085.

7. Lingzhao Fang, Yanhua Li, Yi Zhang, Dongxiao Sun, Lin Liu, Yuan Zhang, Shengli Zhang. Identification of brachyspina syndrome carriers in Chinese Holstein cattle // Journal of Veterinary Diagnostic Investigation. 2013. Vol. 25. N4. P. 4508-4510.

8. Testoni S., Diana A., Olzi E., Gentile A. Brachyspina syndrome in two Holstein calves // Vet. J. 2008. Vol. 177. P. 144-146.

9. Thomsen B., Horn P., Panitz F., Bendixen E., Petersen AH, Holm LE, Nielsen VH. Agerholm JS, Arnbjerg J., Bendixen CA missense mutation in the bovine SLC35A3 gene, encoding a UDP-N- acetylgucosamine transporter, causes complex vertebral malformation // Genome Res. 2006. Vol. 16. P. 97-105.

10. VanRaden P.M., Olson K.M., Null D.J., Hutchison J.L. Harmful recessive effects on fertility detected by absence of homozygous haplotypes // J. Dairy Sci. 2011. Vol. 94. P. 6153-6161.

11. Catalog of manufacturing bulls of the OJSC “Head Center for the Reproduction of Farm Animals” / ed. Yeskina G.V. Bykovo. - 2010 .-- 36 p.

Claims (1)

A method for diagnosing a mutant allele that causes a short spine or brahispin in cattle, including DNA extraction from biological material, real-time polymerase chain reaction using a reaction mixture with BY, a cycling process in which denaturation, annealing of primers and synthesis of a new DNA chains occur at a temperature of 64 ° C, during the first cycling in 10 repetitions, during the second cycling in 30 repetitions, then the reaction results are evaluated: BY is not detected when positive Yelnia channel Green value and a negative value for the channel Yellow, BY positive value detected at the channels Green and Yellow.

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