WO2024252033A1

WO2024252033A1 - Thermo phage test kit

Info

Publication number: WO2024252033A1
Application number: PCT/EP2024/065993
Authority: WO
Inventors: Pim Van Hee; Paulus Petrus DE WAAL; Laurens Leendert HANEMAAIJER; Colleen Mary MCMICHAEL; Ryan RAGLAND
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2023-06-09
Filing date: 2024-06-10
Publication date: 2024-12-12
Anticipated expiration: 2025-12-09
Also published as: EP4724604A1

Abstract

The invention relates to the field of molecular diagnostics, more specifically to a kit for the detection and quantification of thermophilic bacteriophages in a dairy sample.

Description

Thermo phage test kit

The presence of bacteriophages, interchangeably called phages herein, in industrial dairy environment fermentations is generally considered to negatively impact production, with phage infection affecting the rheological and textural properties of the end product. It has been postulated that the main source of new phages entering a product line is raw milk. The first step in efforts to solve this problem is usually the detection and/or quantification of the phages in question. Currently, phage detection is essential to confirm that fermentation slow-down or failure has indeed been caused by the presence of phages but due to the traditionally used methods, remains retrospective.

At present phage detection and quantification is done by using a classical overlay-method or performing an acidification-assay, the former supplying information on phage levels expressed as plaque-forming units (PFU) per millilitre (mL), the latter supplying information on the effect of the phage titres present in a dairy sample on acidification of the strains, which is the primary performance criterium in a dairy fermentation process. In this overlay technique the phage is allowed to propagate in a confluent lawn of bacterial host cells immobilized in a thin and soft layer of top agar, in which a circular transparent area of lysed cells (i.e., the 'plaque') will develop, resulting from a series of phage-infection, phage-multiplication, phage-liberation chain reaction events. However, the effectiveness of the plaque assay to monitor phage is dependent not only upon the phage and the bacterial strain; plaque formation is highly influenced by the (biological) physical and chemical conditions. Accordingly, when propagating phage in a suboptimal environment, plaques may fail to appear. In the acidification-assay a phage is allowed to propagate in liquid medium with a bacterial host and compared with a control only containing liquid medium with the bacterial host. From both, pH is monitored with a pH- indicator or pH-probe. If pH is affected, i.e., pH remains higher compared to control this indicates enough virulent phages were present to affect the culture. Also, this method has its disadvantages and the effectiveness for measuring a pH effect caused by phages depends on (biological) physical and chemical conditions in the liquid medium. When performing an acidification assay under sub-optimal conditions phages can be present without affecting pH, leading to a false negative result. Both methods are time-consuming and labour-intensive. Therefore, only retrospectively such methods establish a phage was causing the fermentation failure rather than enable preventive actions for a dairy producer to suppress the phage titers from causing these failures. Fermentation failures come with a serious economic loss for the dairy producer because of downgrading to less valuable products.

Therefore, a clear need exists for a fast test to detect, classify and quantify phages in a dairy process. This enables the dairy producer to take immediate corrective actions such as heat treatment, apply proper sanitation procedures, or introduce another phage-unrelated rotation culture to suppress the risen phage levels. Currently, for a wide variety of food applications, real-time quantitative polymerase chain reaction (rt-qPCR, or simply called qPCR) has emerged as a method of choice to identify and quantify microbe (contaminant) species due to its rapid and sensitive identification capabilities. Furthermore, recent advances in DNA Polymerase technology (such as polymerase Sso7d- fusion polymerase SsoAdvanced™, Biorad) and optimization of reaction conditions (e.g. intensity and stability of fluorophores such as SYBR-related dyes), opened up possibilities for a fast real-time quantitative polymerase chain reaction protocol (results within one hour) and direct real-time quantitative polymerase chain reaction on matrices containing known PCR-inhibitors without elaborate DNA extraction protocols or samples processing.

In summary, the criteria for a dairy producer to use a phage test kit for fast decision making or point-of-care (POC) test would be the following:

(A) detection of at least one, preferably at least 2, 3 or 4 and most preferably all phages within the prevalent subgroups and relevant to the dairy industrial process which would call for conserved oligonucleotide primers, and optionally a probe, for each subgroup of phages. Preferably, such qPCR assay is multiplex comprised of multiple primer (and probe) sets part of a, preferably lyophilized PCR reaction mixture. Each set of suitable primers and optionally probes need to be directed to conserved genomic regions in the phage DNA to allow for specific detection of each subgroup. The term conserved as used in this context refers to genomic regions in different phages having preferably at least about 90% nucleotide identity, more preferably at least about 95% identity and most preferably at least about 98% identity.

(B) a sensitive test with at least one and preferably at least two of the next features: a quantification efficiency, preferably between 90-110%, linear standard curve (R2 > 0.980), high precision between experiments, consistency across replicate experiments, no primer dimers and a wide dynamic range detecting bacteriophages at least at the same level and preferably below the detection limit of the overlay assay (LOD) < 10³ PFU/ml). The quantification efficiency can be determined by applying the developed qPCR assay on a dilution series of the target DNA with at least three concentrations thereof, preferably diluted ten-fold. The determined Cq values for the dilution series are plotted against the concentration or dilution factor of the target DNA on a logarithmic scale. Through these data points a linear regression curve is generated and the slope of the trend line is calculated. The qPCR efficiency can be calculated using the equation: Efficiency= (I0^(-1/Slope)’¹)*100. Preferably, qPCR efficiencies range from 90% to 110%. And the features of each assay (each primer and probe set to detect each subgroup) in the multiplex assay primer are preferably not influenced by the other assays in the multiplex assay, e.g., primers (and probes) of the different assays are analysed for possible interactions of forming heterodimers with DNA analysis software, and the qPCR efficiency of the multiplex assay is determined by adding multiple target DNA templates to multiple assays. Preferably, the different specific assays for each subgroup result in similar Cq values (± <1 Cq value) when testing a dilution series for further quantification of phage levels.

(C) fast protocol without extensive pre-treatment or DNA purification steps, e.g,. only dilution of milk, allowing for results within about one or two hours.

Since dairy matrices are known to contain compounds inhibiting efficient PCR, especially for reliable quantification, the use of a robust polymerase-primer reaction mixture seems to be a prerequisite for the success of such a kit in the dairy environment. PCR detection of dairy phage subgroups in (processed) dairy samples has been reported in the prior art, even in a multiplex manner (i.e. multiple primer pairs (with optionally probes) targeting a range of distinct phage species in one dairy sample. Labrie and Moinaeu (2000, Appl Environ Microbiol. Vol. 66: pp. 987-994) set up a multiplex PCR assay to detect c2, 936 and P335 subgroups of lactococcal phages in one PCR reaction using whey (powders) as dairy sample. The detection was based on primer design yielding different sized amplicons for each subgroup making interpretation of the presence of each phage group in the sample possible with standard gel electrophoresis. However, the assay purely gives a qualitative result of the presence of a certain phage group or phage groups in the dairy process, but no quantification of phage titers which is needed to indicate the severity of the actual phage problem. Binetti et al. (2005, Appl Environ Microbiol. 71: 6096-6103) developed a PCR detection method for Streptococcus thermophilus phages based on targeting VR2, a variable region of the antireceptor gene claimed by authors to be conserved in all S. thermophilus phages. The assay worked directly on milk samples without pre-treatment or need for DNA purification steps spiked with S. thermophilus phages and showed a detection limit of 10⁵ PFU/ ml. Again, the assay developed only gave a qualitative result here. Similarly, as Labrie and Moineau, Quiberoni et al. (2006, J. Dairy Sci. 89: 3791-3799) developed a multiplex PCR assay for the detection of S. thermophilus cos and pac phages with one PCR reaction. However, this assay was conducted only on phage lysates for typing isolated phages from infected samples and no quantitative results were given. The phage titers determined by classical overlay assay across whey samples ranged from almost 7.5x10² to 3x10⁵ PFU/mL. W02006/136640 discloses a multi-PCR assay to detect phages virulent against Lactobacillus, Lactococcus (c2, 936, P335) and Streptococcus (cos) based on fragment size, but here also no quantification of phage titers was determined. A similar observation can be made for Ali et al. (2014, African J Microbiology research, 8: 2598-2603) in which conserved primers for streptococcal cos and pac phages were developed for a multiplex PCR on phages isolated from yoghurt samples.

Prior art on the development of real-time quantitative polymerase chain reaction protocols for the quantification of phage titers includes Del Rio et al. (2008, Appl Environ Microbiol., 74: 4779-4781) who developed a TaqMan-based (i.e. use of fluorophore-labelled probes) qPCR assay to detect and quantify cos and pac phages directly in artificially spiked ten fold-diluted skimmed milk samples. For both cos and pac assays, one primer-probe set was added to the qPCR assay. The LOD of the developed assays by Del Rio et al. (2008) seemed higher than for the standard plaque assay which is reportedly 10³ PFU/mL observing the supplied data. One microliter of an artificially ten-fold diluted spiked milk sample ranging from 10³ to 10⁹ PFU/mL of cos or pac phage was used a template in the qPCR reaction (total volume 20 microliters). The lowest amount detected for the pac assay seemed to be 10² PFU/reaction (or per microliter of sample) with a Cq value of around 30. This translates to a limit of detection of 10⁵ PFU/mL for the pac assay. For the cos assay the lowest amount detected was 10 PFU/reaction with a Cq value of around 30. This translates to a limit of detection of 10 PFU/mL for the cos assay.

Verreault et al. (2011, Appl Environ Microbiol. 77: 491-497) displayed results of a real-time quantitative polymerase chain reaction protocol to quantify lactococcal 936 and C2 phages in surface or air swab samples from a dairy plant. In this assay, LOD and specificity of the assays was tested and found satisfactory. However, samples analyzed (swabs collected in water with Tween) are relevant for phage management in dairy industry, they are not typical such as milk or whey. Those typical matrices are the most challenging matrices because of their inhibiting substances for the real-time quantitative polymerase chain reaction. Therefore, the robustness of the assays by Verreault et al. (2011) on milk matrices remained elusive. Furthermore, no results on PCR efficiency or limit of quantification were presented in that study.

Ly-Chatain et al. (2011, Int J Microbiol.: 594369) presented a qPCR protocol for detecting c2, 936, and P335 lactococcal phages in whey and raw milk samples. Although the developed protocols showed a sufficient LOD related to the overlay assay (10² PFU/mL) and reasonable qPCR efficiencies (94-98%), the protocol included an extraction protocol to isolate phage DNA from whey and milk samples, thereby removing PCR-inhibiting compounds from the dairy matrix. The extraction protocol included steps, such as using a microcentrifuge and spinning at high gravity force (5000 g), using isopropanol and ethanol to precipitate the phage DNA and using reagents from a DNA isolation kit, which are complicating the proposed protocol to be executed at a dairy customer, which do not have these types of equipment or expertise. Furthermore, the need for specialized molecular biology grade reagents come with added cost to a commercial kit making it less attractive for the dairy customer.

Similarly, Muhammed et al. (2017, PLoS One., 12: e0174223) developed a phage detection /quantification protocol based on multiplex rt-qPCR for c2, 936 and P335 lactococcal phages and additionally Leuconostoc phages with sufficient LOD and qPCR efficiencies for relevant dairy samples. However, similarly as for Ly-Chatain et al. (2011) for sample pre-treatment an elaborate phage DNA purification protocol with multiple steps including specialized molecular DNA extraction or pre-amplification kits is necessary prior to conducting the actual qPCR and obtaining the results. Especially, an overnight Dnase-I treatment prohibits the use of this protocol as a fast test or POC test for fast decision making at a dairy plant. The perspective of Muhammed et al. (2017) was actually more to develop such a protocol for high throughput purposes and retrospective analysis of phage dynamics within process streams in a dairy plant.

For setting up a real-time quantitative method for detection of phage DNA one has to consider the different most prevalent phage subgroups in the dairy environment. The lytic 936, c2 phages and the lysogenic P335 phages cause the most pressing concern for Lactococcus cells in a starter culture. Recently, a test has become available for these mesophilic phages (see WO 2020212376A1). For thermophilic Streptococcus cells in the starter cultures, virulent phages are subdivided based on mode of DNA packaging in the isometric head of the phage, namely the Moineauvirus (interchangeably called cos, herein) and Brussowvirus (interchangeably called pac, herein) Streptococcus thermophilus phages. In recent years two more streptococcal phage groups were identified, i.e., 5093 (Mills et al., 2011, International Dairy Journal, vol. 21, pp. 963-969) and 987 (McDonnell et al., 2016, Applied and Environmental Microbiology, Volume 82, pp. 5153-5165) phages. Altogether, there is a need for an easy-to-use, fast, robust test for these thermophilic phages to accurately quantify their concentrations in dairy samples and to allow for immediate corrective actions in the dairy process. Furthermore, the test needs be able to cover the current diversity of thermophilic phages relevant for the dairy space.

Thus, the present invention provides a quantitative amplification kit for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, wherein the kit comprises at least one of:

(a) a primer pair specific for a conserved genomic region of bacteriophage serovar cos,

(b) a primer pair specific for a conserved genomic region of bacteriophage serovar pac, (c) a primer pair specific for a conserved genomic region of bacteriophage serovar 5093,

(d) a primer pair specific for a conserved genomic region of bacteriophage serovar 987.

Particularly, the present invention provides a quantitative amplification kit for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, wherein the kit comprises at least:

(b) a primer pair specific for a conserved genomic region of bacteriophage serovar pac, and, optionally,

(c) a primer pair specific for a conserved genomic region of bacteriophage serovar 5093, and/or,

Said quantitative amplification kit detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample comprising steps (a) to (d) above may furthermore comprise:

(e) a second primer pair specific for a conserved genomic region of bacteriophage serovar pac.

Further provided is a method for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, comprising the steps of:

(i) obtaining a dairy sample,

(ii) optionally diluting the obtained dairy sample,

(iii) detecting and quantifying the genomic DNA from bacteriophages specific for Streptococcus thermophilus species in the optionally diluted sample in an amplification reaction using a primer pair and optionally a probe as defined herein.

In a further embodiment, the present invention is directed to a kit as defined herein for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample. Furthermore, the present invention provides the use of one or more of the primers sets and / or probes as defined herein for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample.

The quantitative amplification kit is herein referred to the quantitative amplification kit as disclosed herein, the quantitative amplification kit according to the embodiments of the invention or as the kit.

Examples of suitable amplification techniques wherein the kit can conveniently be used are: polymerase chain reaction (PCR), real-time PCR (RT-real-time qPCR), Isothermal amplification methods (like recombinase polymerase amplification (RPA), loop mediated isothermal amplification (LAMP) or others), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA) or ligase chain reaction. The person skilled in the art knowns these techniques and knows how to perform them. Accordingly, when the quantitative amplification kit according to the embodiments herein is used for PCR, a primer (pair) disclosed herein is to be construed as a PCR primer (pair).

A primer pair specific for a conserved genomic region of a bacteriophage serovar is herein to be construed as that such primer pair is suitable for use in an amplification assay wherein that bacteriophage serovar or even a subgroup within each serovar is exclusively detected. The term "conserved" as used herein thus means highly homologous and relates to a genomic region present in all the phages belonging to a certain serovar in which the DNA sequences of the genomic region are highly similar within a phage serovar, however, distinct from other phage serovars. So, e.g. a conserved region in a phage from serovar 987 is highly homologous within and therefore highly specific for the 987 serovar but is not present in any other thermophilic phage serovar. Also primer pairs designed for these conserved region do not adhere under specific conditions to other OFF targets which could interfere with specificity of the kit, such as DNA from mesophilic phages or lactic acid bacteria. Additionally, primers and probes may comprise 1, 2, 3 or 4 degenerate bases to ensure hybridization to all homologues of the conserved region within a serovar. The term conserved as used in this context refers to genomic regions in different phages (belonging to the same serovar) having preferably at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. The kit is used to determine the presence of certain DNA phages in a dairy sample, i.e., to detect phage DNA. Additionally, the kit is preferably used to quantify and, in the end, establish the level of DNA particles from said phages in a dairy sample, i.e., to quantify phage DNA. The kit can also be used to classify the phages present in dairy sample, and hence the invention also provides a kit for detection, quantification and classification of phage DNA from a lactic acid bacteria-infecting phage in a dairy sample. Phage DNA particles are not the same as plaque forming units (PFU). A PFU is visualized with the overlay assay and is the result of a bacteriophage infecting a host leading to lysis of this host. One PFU is the result of multiple bacteriophages that are released upon lysis of the bacterial host, also referred to as burst size. A test kit of the invention detects and quantifies single bacteriophage particles, one bacteriophage infects one lactic acid bacterium. This implies a 1:1 ratio of bacteriophages with bacterial hosts and thereby a correlation of phage particles with acidification. Like in the overlay assay this number is not affected by the phage, hosts or interaction mechanisms between those two, affecting burst sizes. Therefore, the kit of the invention shows a better correlation to acidification compared to the overlay assay. The kit as claimed herein is a kit which allows fast analysis of a dairy sample. The term fast refers to an analysis time of less than about 2 hours, preferably the result is obtained within about 90 minutes and even more preferably results are available within about 60 minutes. The fast analysis is in sharp contrast to the conventional plaque assay which takes at least about 48 hours. Additionally, the analysis is performed by the dairy (for example cheese) manufacturer himself and does not need the sending of a sample (or samples) as is the case with the overlap assay.

The kit as disclosed herein is used to detect and quantify phages in a dairy sample. More in particular, the dairy sample is obtained from a commercial dairy plant, for example a cheese production plant. A dairy sample which can be tested with a kit according to the invention is for example a starting material for a dairy manufacturer (such as bulk starter media, a bulk starter culture or milk). Alternatively, a dairy sample is an intermediate from a dairy manufacturing process (such as acidified milk). The to be tested dairy sample can also be a waste stream from a dairy manufacturing process (such as whey). Alternatively, the to be tested sample is a finished product (for example cheese or a fermented dairy product such as yogurt). Other examples of a dairy sample which can be tested with a kit as disclosed herein are rinse water or a swab from anywhere in a dairy production process. The dairy sample which is tested with a kit as disclosed herein may be whey, a bulk starter media, a bulk starter cultures, milk, acidified milk, whey powder, rinse water, a swab from dairy processes, cheese or a fermented dairy product. More particularly, the dairy sample which is tested with a kit as disclosed herein is whey, a bulk starter media, a bulk starter cultures, milk, acidified milk, whey powder, rinse water or a swab from anywhere in a dairy production process. As used herein the term "rinse water" refers to the liquid resultant from rinsing a fermentation vesicle after a fermentation cycle or cleaning cycle and forming the start condition in the vat for a next fermentation. If phages are present in there in high levels, it could be quite predictive for fermentation failure.

In some embodiments of the invention, the kit may further comprise a second primer pair specific for a conserved genomic region of bacteriophage serovar pac. The inventors arrived at the surprising finding that one primer pair is not sufficient to detect all members within the bacteriophage serovar pac; a second primer pair is required to cover the entire serovar pac.

Particularly, the present invention is directed to a method and a kit as described herein, wherein:

(a) the conserved genomic region of bacteriophage serovar cos may be the COS_P3681_10_protein encoding gene,

(b) the conserved genomic region of bacteriophage serovar pac for the first primer pair may be the Pac_CM2.1_CHPC1152_018 protein encoding gene,

(c) the conserved genomic region of bacteriophage serovar pac for the second primer pair may be the Pac_CM2.2_CHPC1042_015 protein encoding gene,

(d) the conserved genomic region of bacteriophage serovar 5093 may be the 5093_P0095_15 protein encoding gene, and/or

(e) the conserved genomic region of bacteriophage serovar 987 may be the 987_SW28_018 protein encoding gene.

More particularly, the method and kit as described herein are directed to:

(a) the polynucleotide sequence of the COS_P3681_10_protein encoding gene may have at least about 92% sequence identity to SEQ ID NO: 25,

(b) the polynucleotide sequence of the Pac_CM2.1_CHPC1152_018 protein encoding gene may have at least about 88% sequence identity to SEQ ID NO: 27, (c) the polynucleotide sequence of the Pac_CM2.2_CHPC1042_015 protein encoding gene may have at least about 86% sequence identity to SEQ ID NO: 29,

(d) the polynucleotide sequence of the 5093_P0095_15 protein encoding gene may have at least about 90% sequence identity to SEQ ID NO: 21, and/or

(e) the polynucleotide sequence of the 987_SW28_018 protein encoding gene may have at least about 90% sequence identity to SEQ ID NO: 23.

In further embodiments, the method and kit according to the present invention is directed to:

(a) amplification by the primer pair specific for a conserved genomic region of bacteriophage serovar cos may result in an amplicon of which the sequence has at least about 92% sequence identity to SEQ ID NO:1,

(b) amplification by the first primer pair specific for a conserved genomic region of bacteriophage serovar pac may result in an amplicon of which the sequence has at least about 88% sequence identity to SEQ ID NO:2,

(c) amplification by the second primer pair specific for a conserved genomic region of bacteriophage serovar pac may result in an amplicon of which the sequence has at least about 86% sequence identity to SEQ ID NO:3,

(d) amplification by the primer pair specific for a conserved genomic region of bacteriophage serovar 5093 may result in an amplicon of which the sequence has at least about 90% sequence identity to SEQ ID NO: 4, and/or

(e) amplification by the primer pair specific for a conserved genomic region of bacteriophage serovar 987 may result in an amplicon of which the sequence has at least about 90% sequence identity to SEQ ID NO: 5.

In some embodiments of the invention, the method or kit as described herein is directed to:

(a) the forward and reverse primer of the primer pair specific for a conserved genomic region of bacteriophage serovar cos may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:12 and SEQ ID NO:13, respectively,

(b) the forward and reverse primer of the first primer pair specific for a conserved genomic region of bacteriophage serovar pac may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:15 and SEQ ID NO:16, respectively, (c) the forward and reverse primer of the second primer pair specific for a conserved genomic region of bacteriophage serovar pac may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:18 and SEQ ID NO:19, respectively,

(d) the forward and reverse primer of the primer pair specific for a conserved genomic region of bacteriophage serovar 5093 may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:6 and SEQ ID NO:7, respectively, and/or

(e) the forward and reverse primer of the primer pair specific for a conserved genomic region of bacteriophage serovar 987 may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:9 and SEQ ID NQ:10, respectively.

In some embodiments of the invention, the method or kit may further comprise at least one of:

(a) a polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar cos,

(b) a polynucleotide probe capable of hybridizing to the amplicon produced by the first primer pair specific for a conserved genomic region of bacteriophage serovar pac,

(c) a polynucleotide probe capable of hybridizing to the amplicon produced by the second primer pair specific for a conserved genomic region of bacteriophage serovar pac,

(d) a polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar 5093, and/or

(e) a polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar 987.

Particularly, the method or kit according to the present invention is directed to:

(a) the polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar cos may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:14, (b) the polynucleotide probe capable of hybridizing to the amplicon produced by the first primer pair specific for a conserved genomic region of bacteriophage serovar pac may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:17,

(c) the polynucleotide probe capable of hybridizing to the amplicon produced by the second primer pair specific for a conserved genomic region of bacteriophage serovar pac may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:20,

(d) the polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar 5093 may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:8, and

(e) the polynucleotide probe capable of hybridizing to the amplicon produced by the 987 primer pair specific for a conserved genomic region of bacteriophage serovar pac may comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO: 11.

As used herein, at least about 90% sequence identity may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity.

As used herein, at least about 88% sequence identity may be at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity.

As used herein, at least about 86% sequence identity may be at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity.

If, in the embodiments herein, a probe is used, such probe will typically anneal to a target region in the amplicon and will comprise a reporter dye or a reporter dye and a quencher. Such reporter dye may be a fluorophore. Non limiting examples of a suitable fluorophores are 6-carboxylfluorescein (FAM), hexachloro-fluorescein (HEX), 6-carboxy-4'5'-dichloro-2', 7’- dimethoxyfluorescein (JOE), and tetrachlorofluorescein (TET). Typically, fluorophores are placed at the 5'-end of the probe sequence. Examples of suitable quenchers are tetramethylrhodamine (TAMRA), Dabcyl, Black Hole Quenchers, Qxl quenchers, Iowa Black FQ, Iowa Black RQ. Typically, quenchers are placed at the 3'-end of the probe sequence. Probes may additionally comprise an internal quencher (e.g., ZEN) which further reduces background signal and improves specific signal. A probe with an internal quencher in combination with a 3'-quencher is called a double-quenched probe. Probes may also comprise locked nucleic acids (LNA). In one embodiment, a probe is a double-quenched probe optionally further comprising LNA. In one embodiment, a probe is a double-quenched probe comprising Iowa Black FQ, ZEN and optionally comprising LNA. A well-known example of a real-time PCR technique which makes use of a probe is TaqMan. The person skilled in the art knows amplification techniques using probes and knows how to perform these. Preferably, such technique is performed as set forward in the examples herein.

If, in the embodiments herein, a DNA binding dye is used, such DNA binding dye may be any useful dye and preferably is a dimeric dye such as, but not limited to, EvaGreen. Alternatively, the DNA binding dye may be a monomeric dye such as SYBR-Green or any other suitable asymmetrical cyanic dye.

In some embodiments of the invention, the kit may be a real-time qPCR kit, preferably a multiplex real time qPCR kit. The person skilled in the art known this technique and knows how to perform it. Preferably, the technique is performed as set forward in the examples herein.

Typically, an amplification kit such as a qPCR kit as disclosed herein does not only comprise a primer set but may also comprise at least one other compound including but not limited to a DNA polymerase, dNTPs, a bivalent metal ion such as manganese or magnesium, for example magnesium chloride or magnesium sulphate, a probe, and/or DNA binding dye. These other compounds are known to the person skilled in the art and the person skilled in the art knows what concentrations of these compounds should be used and how to optimize the concentrations of these compounds, if required or desired. A DNA polymerase may be an inhibitor tolerant DNA polymerase such as SsoAdvanced (Biorad) or PlatinumTag (Thermofisher), PowerUp (Thermofisher) or BiomemeTaq (Promega).

A kit according to the embodiments of the invention, may further comprise an instruction manual. In an embodiment, the instruction manual comprises instructions to not extract or purify the DNA from the dairy sample. In one embodiment, the instruction manual comprises instructions to dilute the dairy sample, preferably to dilute the dairy sample with water and even more preferably to dilute the dairy sample with tap water. In another embodiment, the dairy sample is diluted at least 10 times, for example by mixing 5 ml of sample with water to a total volume of 50 ml or any other equivalent which results in a dilution of the dairy sample by a factor 10.

In one embodiment, one or more components of the kit are in lyophilized form allowing prolonged storing at ambient temperature.

The present invention furthermore provided is a method for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, comprising the steps of:

(i) obtaining a dairy sample,

(ii) optionally diluting the obtained dairy sample, and

(iii) testing the optionally diluted sample with a kit according to the embodiments of the invention as described herein.

Particularly provided is a method for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, comprising the steps of:

(i) obtaining a dairy sample,

(ii) optionally diluting the obtained dairy sample, and

(iii) detecting and quantifying the genomic DNA from bacteriophages specific for Streptococcus thermophilus species in the optionally diluted sample in an amplification reaction using a primer pair and optionally a probe as defined in any one of the embodiments herein.

The dairy sample can be any of the dairy samples which is described herein in the context of the kit. Typically, a dairy sample is taken at a dairy manufacturer such as a cheese or yogurt manufacturer. The sample may be taken at a cheese manufacturer. The cheese manufacturer may produce cheese on large scale, i.e., a manufacturer which produces at least about 3000 kg cheese per year. A sample may be taken from a batch or fermentation vat or fermentation vessel comprising at least about 50 litres of material. Yet another source of the sample is a sample from a(n) (original) pack size of at least about 10 kg of powder, for example whey powder. The method may be performed at the dairy manufacturer, i.e., the sample does not have to be transported to a test lab outside of the dairy factory. Samples taken in step (i) may be whey, a bulk starter media, a bulk starter cultures, milk, acidified milk, whey powder, rinse water, a swab from dairy processes, cheese or a fermented dairy product. The obtained sample may be tested as such, for example rinse water can be tested as such and does not need a dilution step. Also, after dissolving whey powder in water at an appropriate concentration, the whey powder sample can be tested as such and does not need a dilution step. Also, after processing a swab sample in water, the sample can be tested as such and does not need a dilution step. Some samples may need to be diluted, such as whey, milk, acidified milk, a fermented dairy product or bulk starter broth or media. In optional step (ii) the sample may be diluted with water such as tap water, distilled water, doubledistilled water or molecular grade water (e.g., MilliQ). In one embodiment, the dairy sample is diluted with tap water. Buffers which are qPCR compatible can also be used as a means for diluting the dairy sample.

Accordingly, in the methods according to the embodiments as described herein, the dairy sample from step (i) may be obtained from a dairy production batch of at least about 50 litres or from a pack size of at least about 10 kg of powder. The dairy sample may be whey, a bulk starter media, a bulk starter cultures, milk, acidified milk, whey powder, rinse water, a swab from dairy processes, cheese or a fermented dairy product.

The dairy sample may be diluted at least ten-fold, meaning that x ml sample is diluted such as to end at xO ml of diluted sample (for example 5 ml sample being diluted with help of 45 ml water to a total volume of 50 ml). Such a dilution can easily be performed by the factory worker by using a scoop to hold 5 mL and put it into a tube which has a visible mark at 50 ml allowing an easy dilution step. Another option would be that the factory worker uses a micropipette, which pipettes a fixed volume, or a pastette to pipette 20 microliters of a dairy sample to a 5 mL tube containing 2.48 mL of water, in this way diluting the sample 125-fold. Subsequently, 20 to 100 microliters of the diluted sample may be transferred to a reaction vessel with freeze-dried "phage test reaction mixture" with e.g., a pastette or micropipette. The to be interrogated reaction vessels are transferred to a suitable amplification device such as a qPCR cycler with for example a fluorescence reader such as Biorad CFX system or a suitable mobile device such as three9™ (Biomeme).

Conveniently, a method according to the present invention as described herein uses a portable device in step (iii). The portable device may be a portable device with a display on which the phage risk level is displayed, i.e., the portable device translates the results of the analysis into an advice for the dairy manufacturer, for example to use another rotation of lactic acid bacteria. Particularly, the method may be performed using a portable device for performing the qPCR analysis.

The method according to the invention as described herein may comprise a step wherein DNA is extracted and/or purified from the sample or may not comprise a step wherein DNA is extracted and/or purified from the sample.

The method according to the invention as disclosed herein may be performed such that step (iii) of said method is completed within about 2 hours, such as within about 90 minutes and preferably within about 60 minutes, after obtaining the dairy sample.

Furthermore, the present invention provides the use of a kit as defined herein for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample.

Furthermore, the present invention provides the use of one or more of the primers sets and/or probes as defined herein for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample.

Figure 1 further shows, schematically, a particular embodiment of a device for quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, which comprises:

(i) a sensor 105 to sense a value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in a dairy sample reacting to at least:

(d) a primer pair specific for a conserved genomic region of bacteriophage serovar 987,

(ii) a computing system 200, such as disclosed in regard of Figure 3, configured to, as a function of the sensed value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in the dairy sample, provide a quantity of genomic DNA from said bacteriophages specific for Streptococcus thermophilus species, and

(iii) a computer interface 110, configured to provide the computed quantity of genomic DNA.

The sensor 105 can correspond to any type of sensor used to measure a reaction of a dairy sample to at least:

Such a sensor 105 is adapted to the detection of a physical, chemical and/or biological parameter which is representative of a reaction, and to provide a value representative of this reaction.

Such a sensor 105 may use image recognition techniques, gel electrophoresis, sequencing, or other molecular biology methods. Such a sensor 105 may correspond to a sensor routinely used for PCR testing. Such a sensor may be configured to detect a probe activation of a probe disclosed herein.

The output of the sensor 105 is a raw value of a parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in the dairy sample. This value is then processed, by the computing device 200, to provide a value representative of the presence of bacteriophages specific for Streptococcus thermophilus species in the dairy sample.

The sensor 105 and the computing system 200 are linked by a communication link, which can be of any type. In particular embodiments, the sensor 105 and the computing system 200 are part of a unitary device such as a PCR device, while in other embodiments, the sensor 105 and the computing system 200 are distributed. In the latter embodiments, the sensor 105 and the computing system 200 may be linked via the Internet, for example, or within a local area network. Several such variants are disclosed in relation to Figure 3. Figure 3 represents a block diagram that illustrates an example computer system 200 with which an embodiment of the present invention may be implemented. In the example of Figure 3, a computer system 205 and instructions for implementing the disclosed technologies in hardware, software, or a combination of hardware and software, are represented schematically, for example as boxes and circles, at the same level of detail that is commonly used by persons of ordinary skill in the art to which this disclosure pertains for communicating about computer architecture and computer systems implementations.

The computer system 205 includes an input/output (IO) subsystem 220 which may include a bus and/or other communication mechanism(s) for communicating information and/or instructions between the components of the computer system 205 over electronic signal paths. The I/O subsystem 220 may include an I/O controller, a memory controller and at least one I/O port. The electronic signal paths are represented schematically in the drawings, for example as lines, unidirectional arrows, or bidirectional arrows.

At least one hardware processor 210 is coupled to the I/O subsystem 220 for processing information and instructions. Hardware processor 210 may include, for example, a general-purpose microprocessor or microcontroller and/or a special-purpose microprocessor such as an embedded system or a graphics processing unit (GPU) or a digital signal processor or ARM processor. Processor 210 may comprise an integrated arithmetic logic unit (ALU) or may be coupled to a separate ALU.

Computer system 205 includes one or more units of memory 225, such as a main memory, which is coupled to I/O subsystem 220 for electronically digitally storing data and instructions to be executed by processor 210. Memory 225 may include volatile memory such as various forms of random-access memory (RAM) or other dynamic storage device. Memory 225 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 210. Such instructions, when stored in non-transitory computer-readable storage media accessible to processor 210, can render computer system 205 into a special-purpose machine that is customized to perform the operations specified in the instructions.

Computer system 205 further includes non-volatile memory such as read only memory (ROM) 230 or other static storage device coupled to the I/O subsystem 220 for storing information and instructions for processor 210. The ROM 230 may include various forms of programmable ROM (PROM) such as erasable PROM (EPROM) or electrically erasable PROM (EEPROM). A unit of persistent storage 215 may include various forms of non-volatile RAM (NVRAM), such as FLASH memory, or solid-state storage, magnetic disk, or optical disk such as CD-ROM or DVD- ROM and may be coupled to I/O subsystem 220 for storing information and instructions. Storage 215 is an example of a non-transitory computer-readable medium that may be used to store instructions and data which when executed by the processor 210 cause performing computer-implemented methods to execute the techniques herein.

The instructions in memory 225, ROM 230 or storage 215 may comprise one or more sets of instructions that are organized as modules, methods, objects, functions, routines, or calls. The instructions may be organized as one or more computer programs, operating system services, or application programs including mobile apps. The instructions may comprise an operating system and/or system software; one or more libraries to support multimedia, programming or other functions; data protocol instructions or stacks to implement TCP/IP, HTTP or other communication protocols; file format processing instructions to parse or render files coded using HTML, XML, JPEG, MPEG or PNG; user interface instructions to render or interpret commands for a graphical user interface (GUI), command-line interface or text user interface; application software such as an office suite, Internet access applications, design and manufacturing applications, graphics applications, audio applications, software engineering applications, educational applications, games or miscellaneous applications. The instructions may implement a web server, web application server or web client. The instructions may be organized as a presentation layer, application layer and data storage layer such as a relational database system using structured query language (SQL) or no SQL, an object store, a graph database, a flat file system or other data storage.

Computer system 205 may be coupled via I/O subsystem 220 to at least one output device 235. In one embodiment, output device 235 is a digital computer display or Human Machine Interface. Examples of a display that may be used in various embodiments include a touchscreen display or a light-emitting diode (LED) display or a liquid crystal display (LCD) or an e-paper display. Computer system 205 may include other type(s) of output devices 235, alternatively or in addition to a display device. Examples of other output devices 235 include printers, ticket printers, plotters, projectors, sound cards or video cards, speakers, buzzers or piezoelectric devices or other audible devices, lamps or LED or LCD indicators, haptic devices, actuators, or servos.

At least one input device 240 is coupled to I/O subsystem 220 for communicating signals, data, command selections or gestures to processor 210. Examples of input devices 240 include touchscreens, microphones, still and video digital cameras, alphanumeric and other keys, keypads, keyboards, graphics tablets, image scanners, joysticks, clocks, switches, buttons, dials, slides.

Another type of input device is a control device 245, which may perform cursor control or other automated control functions such as navigation in a graphical interface on a display screen, alternatively or in addition to input functions. Control device 245 may be a touchpad, a mouse, a trackball, or cursor direction keys for communicating direction information and command selections to processor 210 and for controlling cursor movement on display 235. The input device may have at least two degrees of freedom in two axes, a first axis (e.g., x) and a second axis (e.g., y), that allows the device to specify positions in a plane. Another type of input device is a wired, wireless, or optical control device such as a joystick, wand, console, steering wheel, pedal, gearshift mechanism or other type of control device. An input device 240 may include a combination of multiple different input devices, such as a video camera and a depth sensor.

In another embodiment, computer system 205 may comprise an Internet of things (loT) device in which one or more of the output device 235, input device 240, and control device 245 are omitted. Or, in such an embodiment, the input device 240 may comprise one or more cameras, motion detectors, thermometers, microphones, seismic detectors, other sensors or detectors, measurement devices or encoders and the output device 235 may comprise a special-purpose display such as a single-line LED or LCD display, one or more indicators, a display panel, a meter, a valve, a solenoid, an actuator or a servo.

Computer system 205 may implement the techniques described herein using customized hard-wired logic, at least one ASIC or FPGA, firmware and/or program instructions or logic which when loaded and used or executed in combination with the computer system causes or programs the computer system to operate as a special-purpose machine. According to one embodiment, the techniques herein are performed by computer system 205 in response to processor 210 executing at least one sequence of at least one instruction contained in main memory 225. Such instructions may be read into main memory 225 from another storage medium, such as storage 215. Execution of the sequences of instructions contained in main memory 225 causes processor 210 to perform the process steps described herein. In alternative embodiments, hard-wired circuitry may be used in place of or in combination with software instructions.

The term "storage media" as used herein refers to any non-transitory media that store data and/or instructions that cause a machine to operate in a specific fashion. Such storage media may comprise non-volatile media and/or volatile media. Non-volatile media includes, for example, optical or magnetic disks, such as storage 215. Volatile media includes dynamic memory, such as memory 225. Common forms of storage media include, for example, a hard disk, solid state drive, flash drive, magnetic data storage medium, any optical or physical data storage medium, memory chip, or the like.

Storage media is distinct from but may be used in conjunction with transmission media. Transmission media participates in transferring information between storage media. For example, transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus of I/O subsystem 220. Transmission media can also take the form of acoustic or light waves, such as those generated during radio-wave and infra-red data communications.

Various forms of media may be involved in carrying at least one sequence of at least one instruction to processor 210 for execution. For example, the instructions may initially be carried on a magnetic disk or solid-state drive of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a communication link such as a fiber optic or coaxial cable or telephone line using a modem. A modem or router local to computer system 205 can receive the data on the communication link and convert the data to a format that can be read by computer system 205. For instance, a receiver such as a radio frequency antenna or an infrared detector can receive the data carried in a wireless or optical signal and appropriate circuitry can provide the data to I/O subsystem 220 such as place the data on a bus. I/O subsystem 220 carries the data to memory 225, from which processor 210 retrieves and executes the instructions. The instructions received by memory 225 may optionally be stored on storage 215 either before or after execution by processor 210.

Computer system 205 also includes a communication interface 260 coupled to bus 220. Communication interface 260 provides a two-way data communication coupling to network link(s) 265 that are directly or indirectly connected to at least one communication network, such as a network 270 or a public or private cloud on the Internet. For example, communication interface 260 may be an Ethernet networking interface, integrated -services digital network (ISDN) card, cable modem, satellite modem, or a modem to provide a data communication connection to a corresponding type of communications line, for example an Ethernet cable or a metal cable of any kind or a fiber-optic line or a telephone line. Network 270 broadly represents a local area network (LAN), wide-area network (WAN), campus network, internetwork, or any combination thereof. Communication interface 260 may comprise a LAN card to provide a data communication connection to a compatible LAN, or a cellular radiotelephone interface that is wired to send or receive cellular data according to cellular radiotelephone wireless networking standards, or a satellite radio interface that is wired to send or receive digital data according to satellite wireless networking standards. In any such implementation, communication interface 260 sends and receives electrical, electromagnetic, or optical signals over signal paths that carry digital data streams representing various types of information.

Network link 265 typically provides electrical, electromagnetic, or optical data communication directly or through at least one network to other data devices, using, for example, satellite, cellular, Wi-Fi, or BLUETOOTH technology. For example, network link 265 may provide a connection through a network 270 to a host computer 250.

Furthermore, network link 265 may provide a connection through network 270 or to other computing devices via internetworking devices and/or computers that are operated by an Internet Service Provider (ISP) 275. ISP 275 provides data communication services through a world-wide packet data communication network represented as Internet 280. A server computer 255 may be coupled to Internet 280. Server 255 broadly represents any computer, data center, virtual machine, or virtual computing instance with or without a hypervisor, or computer executing a containerized program system such as DOCKER or KUBERNETES. Server 255 may represent an electronic digital service that is implemented using more than one computer or instance and that is accessed and used by transmitting web services requests, uniform resource locator (URL) strings with parameters in HTTP payloads, API calls, app services calls, or other service calls. Computer system 205 and server 255 may form elements of a distributed computing system that includes other computers, a processing cluster, server farm or other organization of computers that cooperate to perform tasks or execute applications or services. Server 255 may comprise one or more sets of instructions that are organized as modules, methods, objects, functions, routines, or calls. The instructions may be organized as one or more computer programs, operating system services, or application programs including mobile apps. The instructions may comprise an operating system and/or system software; one or more libraries to support multimedia, programming or other functions; data protocol instructions or stacks to implement TCP/IP, HTTP or other communication protocols; file format processing instructions to parse or render files coded using HTML, XML, JPEG, MPEG or PNG; user interface instructions to render or interpret commands for a graphical user interface (GUI), command-line interface or text user interface; application software such as an office suite, Internet access applications, design and manufacturing applications, graphics applications, audio applications, software engineering applications, educational applications, games or miscellaneous applications. Server 255 may comprise a web application server that hosts a presentation layer, application layer and data storage layer such as a relational database system using structured query language (SQL) or no SQL, an object store, a graph database, a flat file system or other data storage.

Computer system 205 can send messages and receive data and instructions, including program code, through the network(s), network link 265 and communication interface 260. In the Internet example, a server 255 might transmit a requested code for an application program through Internet 280, ISP 275, local network 270 and communication interface 260. The received code may be executed by processor 210 as it is received, and/or stored in storage 215, or other non-volatile storage for later execution.

The execution of instructions as described in this section may implement a process in the form of an instance of a computer program that is being executed and consisting of program code and its current activity. Depending on the operating system (OS), a process may be made up of multiple threads of execution that execute instructions concurrently. In this context, a computer program is a passive collection of instructions, while a process may be the actual execution of those instructions. Several processes may be associated with the same program; for example, opening up several instances of the same program often means more than one process is being executed. Multitasking may be implemented to allow multiple processes to share processor 210. While each processor 210 or core of the processor executes a single task at a time, computer system 205 may be programmed to implement multitasking to allow each processor to switch between tasks that are being executed without having to wait for each task to finish. In an embodiment, switches may be performed when tasks perform input/output operations, when a task indicates that it can be switched, or on hardware interrupts. Time-sharing may be implemented to allow fast response for interactive user applications by rapidly performing context switches to provide the appearance of concurrent execution of multiple processes simultaneously. In an embodiment, for security and reliability, an operating system may prevent direct communication between independent processes, providing strictly mediated and controlled inter-process communication functionality.

In particular embodiments, the device 100 comprises a comprises a quantitative amplification kit for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample such as disclosed herein, said amplification kit being used prior to the sensor 105 and/or the computing system 200.

The computer interface 110 of the device 100 object of the present invention may correspond to a display destined for a user, such as a computer screen as shown in figure 1, or to an interface destined for a machine, such as an API for example.

Figure 2 represents, schematically, a particular embodiment of the method 300 object of the present invention. This method 300 for quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, comprises:

(i) receiving 305, from a sensor, a value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in a dairy sample reacting to at least:

(c) a primer pair specific for a conserved genomic region of bacteriophage serovar 5093, and/or, (d) a primer pair specific for a conserved genomic region of bacteriophage serovar 987,

(ii) computing 310 a quantity of genomic DNA from said bacteriophages specific for Streptococcus thermophilus species, as a function of the sensed value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in the dairy sample, and

(iii) providing 315, upon a computer interface, the computed quantity of genomic DNA.

Particular embodiments of the steps of receiving 305, computing 310 and providing 315 are disclosed in relation to Figures 1 and 3.

The steps of receiving 305, computing 310 and providing 315 may be performed by a processor executing instructions representative of a computer software. In particular variants, at least two of the steps of receiving 305, computing 310 and/or providing 315 are performed by a unitary computer software. In other variants, each of the steps of receiving 305, computing 310 and/or providing 315 are performed by a dedicated computer software.

It should be noted that the present invention also covers computer program products which comprise instructions which upon execution by a computer cause the computer to execute a method such as shown in Figure 2.

It should be noted that the present invention also covers computer-readable storage media storing programming instructions which upon execution by a computer cause the computer to execute a method such as shown in Figure 2.

The present invention is directed to a device for quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, which comprises:

(i) a sensor to sense a value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in a dairy sample reacting to at least:

(b) a primer pair specific for a conserved genomic region of bacteriophage serovar pac, and, optionally, (c) a primer pair specific for a conserved genomic region of bacteriophage serovar 5093, and/or,

(ii) a computing system configured to, as a function of the sensed value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in the dairy sample, provide a quantity of genomic DNA from said bacteriophages specific for Streptococcus thermophilus species, and

(iii) a computer interface, configured to provide the computed quantity of genomic DNA.

Particularly, a device as described above for quantification of genomic DNA according to claim 16, which comprises a quantitative amplification kit for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample according to one of embodiments as described herein.

Particularly, the invention is directed to a method for quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, which comprises:

(i) receiving, from a sensor, a value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in a dairy sample reacting to at least:

(ii) computing a quantity of genomic DNA from said bacteriophages specific for Streptococcus thermophilus species, as a function of the sensed value for a physical, chemical and/or biological parameter representative of the presence of bacteriophages specific for Streptococcus thermophilus species in the dairy sample, and

(iii) providing, upon a computer interface, the computed quantity of genomic DNA.

Furthermore, the present invention is directed to a Computer program product characterized in that it comprises instructions which upon execution by a computer cause the computer to execute a method according to embodiment as above.

Furthermore, the present invention is directed to a Computer-readable storage medium storing programming instructions which upon execution by a computer cause the computer to execute a method according to embodiment as above.

Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined and the described preferences for one of the above aspects of the invention also apply to the other aspects of the invention.

Figures

Figure 1: schematical representation of a particular embodiment of the device (100) object of the present invention, with a sensor (105), a computing system (200), and a computer interface (110).

Figure 2: schematical representation of a first succession of steps of a particular embodiment of the method (300) object of the present invention, with the steps of receiving (305), computing (310) and providing (315).

Figure 3: schematical representation of a particular computing system (200) capable of performing the method object of the present invention, with a server (255), Internet (280), an Internet provider (275), a network (270), a host computer (250), a unit of persistent strorage (215), a read only memory (230), a memory (225), a computer system (205), an input/output subsystem (220), a network link (265), a communication interface (260), a hardware processor (210), an output device (235), an input device (240), and a control device (245).

The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents, and published patent applications, cited throughout this application are hereby incorporated by reference. Examples

Example 1: General methods and materials

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or

Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).

Sequences. The actual sequences are depicted in the sequence listing and as follows (Table 1). Table 1. Description of sequences.

PCR assay design. The PanelPlex-Consensus™ modality of the DNAsoftware® web-based software suite was used to identify the potential oligonucleotides for each assay. Inclusivity (i.e., on-target), and exclusivity and background (i.e., off- target) playlists were specified in the software. In some cases, a representative sequence ("keystone"), with or without a defined region -of- interest, was also specified. Additional chosen settings include Taqman Probe Detection Type, FastCompare Scores Targan Usage, and Biomeme LyoDNA2.0 PCR Kit (0.05 M Na and 0.008 M Mg), with preset values used in other fields. The coverage of high- ranking results was assessed against the inclusivity playlist to determine if further optimizations could be made to the design of the software- recommended oligonucleotide primers and probes before ordering them along with synthetic gene block (gBlock Gene Fragments) templates from Integrated DNA Technologies.

PCR assay validation. A primer concentration matrix, using all combinations of 100, 200, 400, or 800 nM, for each potential target was analysed in a 20-pL PCR using a mastermix with SYBR Green spiked in and 5E+04 copies of the appropriate gene block as template. Primers in their respective concentrations and 200 nM probes for each singleplex in mastermix were evaluated in triplicate with a standard curve comprising a ten-fold dilution series from 3E+06 to 30 copies of the appropriate gene block using the following protocol (95°C 3 minute, 45 cycles of 95°C 1 second, 60°C 15 seconds, and 77°C 25 seconds) with a baseline threshold set at 75 RFU on a Bio- Rad CFX96 real-time PCR thermocycler. Primer and probe concentrations were further optimized in singleplex based upon the standard curve results. Matched and mismatched gene block templates, for the very divergent Cos and Pac phage classes, that represented the full diversity of the appropriate inclusivity playlist were used. Acceptable PCR efficiencies ranged from 90 - 110%. Assay performance was further re-assessed, re-optimized, and/or redesigned in cases where the efficiencies fell outside this acceptance range or where the Cq or the end RFU for 30 copies of template exceeded 37 cycles or 1000 RFU, respectively. Multiplexed assays comprised all the primers and probes, mastermix, and an additional 2 units of Taq polymerase were tested on individual gene block templates separately using the same dilution series as for the singleplex PCR. Efficiency, Cq, and RFU acceptance criteria for multiplexed reactions were the same as for the singleplex PCRs.

Incubation experiments. Inoculum of DS84445-lac^neg and DS84445-lac^neg-pGdh442 strains for the incubation experiments was prepared from precultures. Thereto, cells were harvested from full grown GM17 precultures by centrifugation at 10,670g for 5 minutes at 4°C and washed twice with 50 mM sodium phosphate buffer (pH 6.5 +/- 0.1). Incubation media were prepared based on chemically defined medium (CDM) described by Otto et al. (FEMS Microbiology Letters 16, 69-74, 1983), whereby the following adaptions were made. Depending on the experiment, CDM was made with either potassium phosphate buffer (39 mM, pH 6.5 +/- 0.1) or with citrate-phosphate buffer containing 50 mM citric acid and 100 mM disodium hydrogen phosphate (pH 5.2 +/- 0.1). The CDM media with initial pH of 6.5 and 5.2 were inoculated with concentrated cell suspension obtained from the precultures to a final cell count of 2 x 107 or 2 x 108 per ml, respectively (determined by flow cytometry). Cell counts were measured as described below from which inoculation rate was calculated. Directly after inoculation, the 15 ml CDM was divided in 2 portions. A portion of 13 ml was transferred to 15 ml conical tube (Greiner, Pleidelsheim, Germany) and the other portion of 2 ml was transferred to 10 ml air-tide sealed vials (Crimp-nek N20, Macherey-Nagel, Germany), and both were incubated at 30°C. For each time point samples were prepared in triplicate. The conical tubes were taken directly after inoculation and after different incubation times (24, 48 and 72h), and used for cell counts determination via selective plating or live/dead counting. Vials were stored at - 40°C until further analysis. For each experiment non-inoculated blanks were taken along and incubated under the same conditions. Presented data represent average of biological triplicates. All incubations were performed at 30°C. Microbial analysis. For evaluation of viable cell counting, appropriate dilutions of samples taken from CDM incubations were made in PPS and streaked-out on GM17 agar plates and incubated in an aerobic jar at 30°C for 5 days.

For live/dead cell counting, the number of cells with non-compromised cell membrane (live) and cells with a compromised cell membrane (damaged or dead) was determined using LIVE/DEAD Baclight Bacterial Viability kit (ThermoFisher Scientific, Massachusetts, USA) and analysed using a CytoFLEX Flow Cytometer (Beckman Coulter, California, USA). The CDM samples were diluted 100 or 1,000 times with PPS, and 199 pl of each diluted sample was added into a 96-well F-bottom microtiter plate (Greiner, Frickenhausen, Germany). A staining mixture of 4.5 pl SYTO 9, 4.5 pl propidium iodide (Pl) and 141 pl demi waters was prepared. Cells were stained by adding 1 pl staining mixture to each 96-well F-bottom microtiter plate, which was stored in the dark for 15 minutes at room temperature. The green fluorescent SYTO 9 labelled the nuclei of live cells, while the red-fluorescent PI labelled dead cells or cells with a compromised cell membrane. Number of live, damaged, and dead cells were counted with the flow cytometer at 488 nm to a maximum of 10,000 events. During all measurements the number of damaged cells was less than 1% of the number of live cells.

Example 2: Multiplex Thermophage detection assay

A multiplex Thermophage detection assay is desired that will (1) be able to sensitively detect and cover all relevant bacteriophages of Streptococcus thermophilus (from hereon called thermophages) that have the potential to negatively affect the cheesemaking process in which culture blends comprised of Streptococcus thermophilus are used and their phages can cause issues, (2) be as simple as possible for inexperienced end users to conduct on-site without expensive specialized laboratory equipment, and (3) have a rapid sample- to- answer turnaround time.

Developed was a specific and sensitive (LoD < 30 template copies; 90-110% efficiency range) real-time PCR assay that can be used with diluted milk matrices to shorten turnaround time from 5 days to 1 hour (see Ex. 1). The thermophages required to be detected by the assay are grouped into four classes: 5093, 987, Cos, and Pac. The target regions within the genomes of the bacteriophages to be detected are depicted in Table 2. The primers and probes are depicted in Table 3. The results of the PCR in multiplex are depicted in Table 4.

Table 2. Sequences of the target genes. For more details, see text.

Table 3. Sequences and primers, probes and amplicons, "desc" means description of the sequence, "A" means Amplicaon, "cone" is the concentration in nM, "FP" means forward primer, "RP" means reverse primer, "pr" menas probe, "6-FAM" means fluorophore 6-carboxylfluorescein, ZEN is an internal quencher, lABkFQ is quencer Iowa Black FQ, "+" preceding a base means a locked nucleic acid (LNA). For more details, see text.

The performance of the real-time PCR assay for each of the phage classes was achieved, initially tested in singleplex PCRs that were successful in detecting as few as 30 copies of the target templates within an efficiency range of 91.5-94.8%. In their respective singleplex PCRs, all targets in 10-fold dilution series were detected with Cq values in the range of 15.36 (± 0.17) to 35.44 (± 0.45) and end RFUs in the range of 1114 (± 219) to 5086 (± 451), data not shown.

In multiplex, each target was successfully detected at the 30-copy sensitivity limit, within an efficiency range of 90.1-95.5%. In the multiplex PCRs, all targets in 10-fold dilution series were detected with Cq values in the range of 15.36 (± 0.30) to 35.84 (± 0.37) and end RFUs in the range of 572 (± 193) to 5895 (± 318), see Table 4.

Table 4. Results of multiplex PCR. Depicted are Cq and End RFU means and their standard deviations (SD), as well as linear regression coefficients and efficiency calculations, for a standard curve for each template in multiplexed PCRs. For more details, see text.

Claims

1. A quantitative amplification kit for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, wherein the kit comprises at least:

2. 2. A kit according to claim 1, further comprising a second primer pair specific for a conserved genomic region of bacteriophage serovar pac.

3. A kit according to claim 1 or 2, wherein:

(a) the conserved genomic region of bacteriophage serovar cos is the COS_P3681_10_protein encoding gene,

(b) the conserved genomic region of bacteriophage serovar pac for the first primer pair is the Pac_CM2.1_CHPC1152_018 protein encoding gene,

(c) the conserved genomic region of bacteriophage serovar pac for the second primer pair is the Pac_CM2.2_CHPC1042_015 protein encoding gene,

(d) the conserved genomic region of bacteriophage serovar 5093 is the 5093_P0095_15 protein encoding gene, and/or

(e) the conserved genomic region of bacteriophage serovar 987 is the 987_SW28_018 protein encoding gene.

4. A kit according to claim 3, wherein:

(a) the polynucleotide sequence of the COS_P3681_10_protein encoding gene has at least about 90% sequence identity to SEQ ID NO:25,

(b) the polynucleotide sequence of the Pac_CM2.1_CHPC'l'l52_018 protein encoding gene has at least about 88% sequence identity to SEQ ID NO:27, (c) the polynucleotide sequence of the Pac_CM2.2_CHPC1042_015 protein encoding gene has at least about 86% sequence identity to SEQ ID NO:29,

(d) the polynucleotide sequence of the 5093_P0095_15 protein encoding gene has at least about 90% sequence identity to SEQ ID NO:21, and/or

(e) the polynucleotide sequence of the 987_SW28_018 protein encoding gene has at least about 90% sequence identity to SEQ ID NO:23.

5. A kit according to any one of claims 1 to 4, wherein:

(a) amplification by the primer pair specific for a conserved genomic region of bacteriophage serovar cos results in an amplicon of which the sequence has at least about 90% sequence identity to SEQ ID NO:1,

(b) amplification by the first primer pair specific for a conserved genomic region of bacteriophage serovar pac results in an amplicon of which the sequence has at least about 88% sequence identity to SEQ ID NO:2,

(c) amplification by the second primer pair specific for a conserved genomic region of bacteriophage serovar pac results in an amplicon of which the sequence has at least about 86% sequence identity to SEQ ID NO:3,

(d) amplification by the primer pair specific for a conserved genomic region of bacteriophage serovar 5093 results in an amplicon of which the sequence has at least about 90% sequence identity to SEQ ID NO:4, and

(e) amplification by the primer pair specific for a conserved genomic region of bacteriophage serovar 987 results in an amplicon of which the sequence has at least about 90% sequence identity to SEQ ID NO:5.

6. A kit according to any of claims 1 to 5, wherein

(a) the forward and reverse primer of the primer pair specific for a conserved genomic region of bacteriophage serovar cos comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:12 and SEQ ID NO:13, respectively,

(b) the forward and reverse primer of the first primer pair specific for a conserved genomic region of bacteriophage serovar pac comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:15 and SEQ ID NO:16, respectively,

(c) the forward and reverse primer of the second primer pair specific for a conserved genomic region of bacteriophage serovar pac comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:18 and SEQ ID NO:19, respectively,

(d) the forward and reverse primer of the primer pair specific for a conserved genomic region of bacteriophage serovar 5093 comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:6 and SEQ ID NO:7, respectively, and/or

(e) the forward and reverse primer of the primer pair specific for a conserved genomic region of bacteriophage serovar 987 comprise or consist of a sequence that has at least about 90% sequence identity with SEQ ID NO:9 and SEQ ID NQ:10, respectively.

7. A kit according to any of claims 1 to 6, further comprising at least one of:

8. A kit according to claim 7, wherein:

(a) the polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar cos comprises or consists of a sequence that has at least about 90% sequence identity with SEQ ID NO:14,

(b) the polynucleotide probe capable of hybridizing to the amplicon produced by the first primer pair specific for a conserved genomic region of bacteriophage serovar pac comprises or consists of a sequence that has at least about 90% sequence identity with SEQ ID NO:17,

(c) the polynucleotide probe capable of hybridizing to the amplicon produced by the second primer pair specific for a conserved genomic region of bacteriophage serovar pac comprises or consists of a sequence that has at least about 90% sequence identity with SEQ ID NO:20,

(d) the polynucleotide probe capable of hybridizing to the amplicon produced by the primer pair specific for a conserved genomic region of bacteriophage serovar 5093 comprises or consists of a sequence that has at least about 90% sequence identity with SEQ ID NO:8, and/or

(e) the polynucleotide probe capable of hybridizing to the amplicon produced by the 987 primer pair specific for a conserved genomic region of bacteriophage serovar pac comprises or consists of a sequence that has at least about 90% sequence identity with SEQ ID NO:11.

9. A kit according to any of claims 1 to 8, wherein said kit is a real-time qPCR kit, preferably a multiplex real time qPCR kit.

10. A method for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, comprising the steps of:

(i) obtaining a dairy sample,

(ii) optionally diluting the obtained dairy sample,

(iii) testing the optionally diluted sample with a kit according to any one of claims 1 to 9.

11. A method for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, comprising the steps of:

(i) obtaining a dairy sample,

(ii) optionally diluting the obtained dairy sample,

(iii) detecting and quantifying the genomic DNA from bacteriophages specific for Streptococcus thermophilus species in the optionally diluted sample in an amplification reaction using a primer pair and optionally a probe as defined in any one of claims 1 to 9.

12. A method according to claim 10 or 11, wherein the dairy sample from step

(i) is obtained from a dairy production batch of at least about 50 litres or from a pack size of at least about 10 kg of powder.

13. A method according to any one of claims 10 to 12, wherein the dairy sample is whey, a bulk starter media, a bulk starter cultures, milk, acidified milk, whey powder, rinse water, a swab from dairy processes, cheese or a fermented dairy product.

14. A method according to any one of claims 10 to 13, wherein the method does not comprise a step wherein DNA is extracted and/or purified from the sample.

15. A method according to any one of claims 10 to 14, wherein step (iii) of said method is completed within about 2 hours, preferably within about 90 minutes and more preferably within about 60 minutes, after obtaining the dairy sample.

16. A device for quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, which comprises:

17. A device for quantification of genomic DNA according to claim 16, which comprises a quantitative amplification kit for detection and quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample according to one of claims 1 to 9.

18. A method for quantification of genomic DNA from bacteriophages specific for Streptococcus thermophilus species in a dairy sample, which comprises:

19. Computer program product characterized in that it comprises instructions which upon execution by a computer cause the computer to execute a method according to claim 18.

20. Computer-readable storage medium storing programming instructions which upon execution by a computer cause the computer to execute a method according to claim 18.