Quick and cheap MIRU-VNTR typing of Mycobacterium tuberculosis species complex using duplex PCR

Accepted Manuscript Quick and cheap MIRU-VNTR typing of Mycobacterium tuberculosis species complex using duplex PCR Memona Yasmin, Stéphanie Le Moullec, Rubina Tabassum Siddiqui, Jessica De Beer, Christophe Sola, Guislaine Refrégier PII:

S1472-9792(16)30294-3

DOI:

10.1016/j.tube.2016.10.003

Reference:

YTUBE 1535

To appear in:

Tuberculosis

Received Date: 29 July 2016 Revised Date:

23 September 2016

Accepted Date: 2 October 2016

Please cite this article as: Yasmin M, Le Moullec S, Siddiqui RT, De Beer J, Sola C, Refrégier G, Quick and cheap MIRU-VNTR typing of Mycobacterium tuberculosis species complex using duplex PCR, Tuberculosis (2016), doi: 10.1016/j.tube.2016.10.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Quick

and Cheap MIRU-VNTR Typing of Mycobacterium

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tuberculosis Species Complex Using Duplex PCR

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Authors: Memona Yasmina,b, Stéphanie Le Moullecc, Rubina Tabassum Siddiqui a,

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Jessica De Beer d, Christophe Solac and Guislaine Refrégier c*.

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a

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Health Biotechnology Division, National Institute for Biotechnology and Genetic

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Engineering (NIBGE), P.O.Box# 577, Jhang Road, Faisalabad, Pakistan.

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b

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Islamabad, Pakistan.

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c

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Université Paris‐Saclay, 91198, Gif‐sur‐Yvette cedex, France

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d

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Environment (RIVM), Bilthoven, The Netherlands.

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Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore,

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris‐Sud,

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National Tuberculosis Reference Laboratory, National Institute for Public Health and the

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*

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France, Tel: +33-1691-54-648 ; Fax: +33-1691-56-678 ; E-mail: guislaine.refregier@u-

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psud.fr

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Corresponding author: Guislaine Refrégier, I2BC Bat 400, 91405 Orsay Cedex,

Running Title: Duplex

MIRU-VNTR Typing for TB

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Abstract: While minisatellites are usually typed using capillary sequencers or qiaplex

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systems in developed countries, many low-resource regions cannot afford it. We propose

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an optimized agarose gel electrophoresis method to genotype Mycobacterium

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tuberculosis species complex minisatellites in their standardized format (24 MIRU-

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VNTR). It is based on duplex PCRs combining VNTR loci harboring distinct amplicon

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sizes whatever the repetition number of each locus. This method performs well both on

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DNA extracts of good quality and on thermolysates while reducing both workload and

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reagents costs.

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79 words

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Keywords: MLVA; multiplexing; genotyping.

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Introduction

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Even if Whole Genome Sequencing is in the path for becoming a surveillance tool,

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Mycobacterium tuberculosis species complex (MTC) outbreak surveillance relies still

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today in most of the countries on the standardized typing of 24 minisatellite loci (MIRU-

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VNTR for Mycobacterial Interspersed Repetitive Units-variable Number of Tandem

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Repeats) (Supply et al, 2006) preferentially in combination with spoligotyping (Azé et al,

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2015). The faster protocol to perform 24 MIRU-VNTR-typing is based on gel or capillary

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electrophoresis-based sequencers (Supply et al, 2006), and requires only 6 or 8 PCR per

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sample because of the use of quadri- or triplex PCRs. Even though prices are decreasing,

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fluorescent molecules for capillary sequencing remain relatively costly so that many

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laboratories, especially in resource-limited countries, perform simplex PCR and run them

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on agarose gels using standard electrophoresis devices. As a consequence, they perform

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24 PCR per sample and run them on as many gels. The analysis of 96 samples requires

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around 2 months for a fully trained technician.

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We aimed at proposing duplex PCRs to reduce both workload and reagent costs.

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Materials and methods

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We mined MIRU-VNTR diversity in 1) a publication of reference from Wirth et al.

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(2008), and 2) in the RIVM database (Azé et al, 2015; Sloot et al, 2013). We defined as

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“most common size range” for each MIRU-VNTR locus all amplicon sizes for which the

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two studies had at least two instances each. These ranges were used to pair the duplexes

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with minimal overlap. In case of overlap, we associated loci so that a difference of at

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least 16 bp remained between any paired amplicons.

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DNA samples of the Quality Control studies were extracted using CTAB technique (De

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Beer et al, 2014); they were sent by RIVM at Room Temperature at a concentration of

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100 ng/µL and diluted before use to 10 ng/µL. DNA samples from Pakistan (n=255) were

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extracted by CTAB technique or by thermolysis in central laboratory of Faisalabad. Out

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of the 215 samples extracted by CTAB, 32 samples were stored 1 month as pellets and

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183 samples were directly resuspended in 100 µL Tris EDTA buffer 10mM/1mM. All

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samples extracted by thermolysis (n=40) were obtained by three cycles of boiling-freeze

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and thaw of a loop of solid bacterial culture in 100 µL of Tris EDTA buffer 10mM/1mM,

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followed by centrifugation and removal of cell pellets. All the DNA samples were stored

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at -20°C and shipped under dry ice to the French laboratory for further characterization.

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PCR reactions were performed in 15 µL as described in Le Fleche et al. (2002) using 5 to

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70 ng of purified DNA by adding 2 µL of concentrated or diluted DNA solution, or 2 µL

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of thermolysate. Amplifications were performed with the following final concentrations:

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Tris-HCl (pH=8.75) 20 mM; KCl 10 mM; (NH4)2SO4 10 mM; MgSO4 2 mM; MgCl2 1.5

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mM; Triton 0.1%; dNTP 0.3 µM; Betain 250mM; DMSO 5%; 0.5 U home-made cloned

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Taq DNA polymerase or using GoTaq (Promega); 0.66 or 1 µM of each primer (see

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Results). Amplification program consisted in: 5 min at 94°C; 35 cycles at 94°C 30s, 61°C

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30s, 72°C 1min 30s; 72°C 5 min. Ten microliters (10 µL) of PCR product were loaded on

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a 2% agarose gel no further than 4 wells of a 100bp DNA ladder covering the range 100

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to 1500 bp. All samples were handled with 8 multichannel pipet using tips 4 by 4 to

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reduce chances for erroneous pipetting. The gels were run for 2 hours under 350 V. The

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gels were subsequently stained with ethidium-bromide under standard conditions.

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Reading was performed by two independent readers, one technician or student and one

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researcher. Transfer to digital format occurred simultaneously.

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Results and Discussion

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We aimed at proposing duplex PCRs to reduce both workload and reagent costs.

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We first identified common ranges of each MIRU-VNTR locus using both published data

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from Wirth et al. (2008) and the diversity identified in the RIVM database 2004-2008

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including 3454 isolates (Azé et al, 2015; Sloot et al, 2013). We separated them in 3

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categories according to their length (short, intermediate and long) (Table 1). We

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identified 34 possible pairs coupling VNTR with different size ranges and exhibiting

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differences of at least 16 bp between almost any reported amplicon size. We tested these

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pairs on reference DNA H37Rv. Four pairs were rejected due to failure to amplify DNA

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for one of the two targeted VNTR (ETRC-ETRE; Mtub34-MIRU27; Qub26-Mtub30;

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Qub4156-Mtub30, Supplementary table). The primers’ concentrations for the other

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pairs were adjusted to improve homogeneity between amplification intensities. We

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selected a combination of 12 couples that amplified efficiently both VNTR of the couple

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and covered the 24-MIRU-VNTR panel (Table 2). Table 3 lists the correspondence

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between amplicon sizes and the number of repeats for each VNTR, highlighting standard

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ranges and possible ambiguities.

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We applied this combination to two Quality Control (QC) Studies coordinated by the

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RIVM: QC2 (de Beer et al, 2014) and QC3 (de Beer, unpublished data). The

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corresponding sets are built so as to represent a large diversity of MIRU-VNTR patterns.

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All duplexes amplified correctly except Miru16-Qub11b for which Qub11b failed to

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amplify. This failure was later attributed to an inversion in the concentrations initially set

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up for each VNTR. Later experiments showed good amplification for this duplex too.

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The total number of samples to be repeated in simplex was 56 (8%) and 81 (11.3%) in the

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two QC studies respectively. For 32 and 41 instances out of 720 datapoints (4.4 and

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5.7%), amplicon sizes alone was not sufficient to assign them to one locus or the other,

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but intensity of the amplicon as compared to that of other samples unequivocally assured

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correct interpretation of the assignation of the amplicon to one locus in all but one case

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concerning Mtub21 and Mtub39 duplex. The few errors committed in these QC studies as

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detected by RIVM reference center (n=4 out of 1440 data points, 0,3%) were due to 2

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errors in data transfer from book to file, 1 error in size reading of large fragments, one

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error in picking the sample when repeating it due to absence of result in the first trial.

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Overall, we scored at 100% intralaboratory reproducibility and 93% interlaboratory

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reproducibility in both studies as in both studies two genotyping errors occurred

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concerning each time two non-duplicated samples. This level of quality is far above the

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average for agarose gel method (82 and 76% respectively in QC2) (de Beer et al, 2014).

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The reduction of the number of individual tubes to handle may be responsible for this

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gain in reliability as compared to simplex method.

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We applied the same procedure onto 255 samples from Pakistan (215 CTAB extracted;

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40 thermolysates), some of which have been described in a previous study (Yasmin et al,

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2014). DNA concentrations were highly diverse, between 1 and 765 ng/µL. Most

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concentrated samples were diluted 10 or 20 times to reach concentrations between 5 and

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35 ng/µL.

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Altogether, amplification occurred successfully (Fig. 1A) and amplicon sizes enabled

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easy attribution to one VNTR or the other. In some instances, a single but thicker band

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could be observed and corresponded to a double band for very similar amplicon sizes

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(Fig. 1B, sample 416). In addition, one pair of primers was wrongly assembled, leading

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to patterns that could not be interpreted (n=512). This error was easily detected thanks to

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H37Rv amplicon sizes. The corresponding VNTR were repeated in simplex. Altogether

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5,093 data points could be directly obtained (83%, n=6,144). Repetitions in simplex were

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performed for all failed isolates (n= 1,039) unless insufficient amount of DNA. After 3

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repetitions and 4205 PCR tubes, we obtained 187 samples with complete genotype, 14

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with 23 typed VNTR, 7 with 22 typed VNTR, i.e. 208 with exploitable data (82%,

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n=255; 84% of CTAB-extracted samples n=180 out of 215, and 70% of thermolysates,

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n=28 out of 40) (Supplemental Table 2). Among exploitable data, a higher percentage

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of CTAB-extracted samples were successfully typed at the 24 loci (n=165; 77%) as

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compared to thermolysates (n=22; 55%). The forty-eight samples exhibiting 21 or less

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VNTR data were excluded from further analyses. Altogether, workload corresponded to

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4205 tubes which represents 69% of the number of tubes in case of performing simplex

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analysis without any repetition. This analysis was completed in 10 weeks by a single

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person in charge of the experiments; a second person having controlled data

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interpretation (~1 week workload). As such total workload and reagents costs were

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reduced by at least 30%. Failure to type some isolates may have been caused by poorer

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DNA quality of thermolysates, by lower DNA concentration as observed in a selection of

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failed isolates (isolate 292: 3 ng/µL; isolate 438 1ng/µL) or by degradation during storage

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(22 out of 35 failed CTAB-extracted isolates come from the extraction batch stored as

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pellets).

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Conclusion

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The technique we propose allows speeding up genotyping of Mycobacterium tuberculosis

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complex isolates from which DNA extracts are of good quality. It reduces workload and

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reagent costs by more than 30%. The protocol is not more complex than that of simplex,

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but requires rigor to appropriately pair primers at the advised concentrations. As it

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reduces the number of tubes to handle, it helps reducing errors due to large experiment

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management.

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The same type of duplexes can easily be transferred to other pathogens that are typed

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using mini-satellites.

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Acknowledgements

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We acknowledge University of Paris-Sud financial support. We thank J. Zhang and M.

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Gomgnimbou for help in daily laboratory management, and Gilson International (Villiers

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le Bel, France) for technical support.

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References

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Azé J, Sola C, Zhang J, Lafosse-Marin F, Yasmin M, Siddiqui R, et al. Genomics and Machine Learning for Taxonomy Consensus: The Mycobacterium tuberculosis Complex Paradigm. PLoS One 2015; 10: e0130912.

180 181 182

de Beer JL, Kodmon C, van Ingen J, Supply P, van Soolingen D. Second worldwide proficiency study on variable number of tandem repeats typing of Mycobacterium tuberculosis complex. Int J Tuberc Lung Dis 2014; 18: 594-600.

183 184 185

Le Fleche P, Fabre M, Denoeud F, Koeck JL, Vergnaud G. High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing. BMC Microbiol 2002; 2: 37.

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Sloot R, Borgdorff MW, de Beer JL, van Ingen J, Supply P, van Soolingen D. Clustering of tuberculosis cases based on variable-number tandem-repeat typing in relation to the population structure of Mycobacterium tuberculosis in the Netherlands. J Clin Microbiol 2013; 51: 2427-2431.

190 191 192 193

Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, et al. Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive UnitVariable-Number Tandem Repeat Typing of Mycobacterium tuberculosis. J Clin Microbiol 2006; 44: 4498-4510.

194 195 196

Wirth T, Hildebrand F, Allix-Beguec C, Wolbeling F, Kubica T, Kremer K, et al. Origin, spread and demography of the Mycobacterium tuberculosis complex. PLoS Pathog 2008; 4: e1000160.

197 198 199

Yasmin M, Gomgnimbou MK, Siddiqui RT, Refregier G, Sola C. Multi-drug resistant Mycobacterium tuberculosis complex genetic diversity and clues on recent transmission in Punjab, Pakistan. Infection Genetics and Evolution 2014. 27: 6-14.

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186 187 188 189

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Funding

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Memona Yasmin stay in Université Paris-Sud laboratory was funded by Erasmus [grant number 744079L]. G. Refrégier and Christophe Sola acknowledge recurrent support from CNRS-Université Paris-Sud.

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Conflict of Interest

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We declare no financial or other type of conflict of interest.

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Table 1- Size ranges of MIRU-VNTR amplicons Most frequent MIRU-VNTR

range (according to all explored data)

amplicons

Intermediate amplicons

Long

178-292 230-404 205-481 261-435 149-377 293-350 225-279 218-422 322-472 330-561 283-515 407-677 264-1041 508-561 447-500 643-749 671-777 514-591 766-872 562-817 657-709 598-757 646-699 622-799

212 213

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Amplicons

ETRB ETRC Qub11b Mtub30 Mtub21 Mtub29 Mtub34 Mtub04 ETRA MIRU04(ETRD-1) Mtub39 MIRU40 Qub26 MIRU02 MIRU24 MIRU10 MIRU16 MIRU20 MIRU23 MIRU26 MIRU27 MIRU31-ETRE MIRU39 Qub4156

Size range in

according to Wirth

RIVM database

et. al (2008)

(2004-2008)

178-463 230-520 136-826 261-493 149-890 179-407 171-495 177-575 322-922 253-638 283-979 407-785 264-1485 455-561 447-712 588-855 618-1147 514-591 660-1137 511-919 551-709 545-863 593-752 622-799

121-634 172-578 136-895 261-609 149-890 179-464 171-441 177-728 247-997 253-946 283-1443 407-893 264-1485 455-614 447-712 535-1065 618-988 514-668 607-1137 460-1123 551-762 545-916 593-858 563-1035

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Short

Size range

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Tables

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Table 2- Primer concentrations in duplex reactions

ETR-A

ATTTCGATCGGGATGTTGAT

TCGGTCCCATCACCTTCTTA

1

MIRU 39

CGCATCGACAAACTGGAGCCAAAC

CGGAAACGTCTACGCCCCACACAT

0.66

ETR-B

GCGAACACCAGGACAGCATCATG

GGCATGCCGGTGATCGAGTGG

0.66

Qub 26

GGCCAGGTCCTTCCCGAT

AACGCTCAGCTGTCGGAT

1

3

ETR-C

GACTTCAATGCGTTGTTGGA

GTCTTGACCTCCACGAGTGC

0.66

MIRU 20

TCGGAGAGATGCCCTTCGAGTTAG

GGAGACCGCGACCAGGTACTTGTA

0.66

GCGCGAGAGCCCGAACTGC

GCGCAGCAGAAACGTCAGC

0.66

GCCACCTTGGTGATCAGCTACCT

0.66

TACTCGGACGCCGGCTCAAAAT

0.66

MIRU4 4

5

6

7

(ETRD-1)

GTTCTTGACCAACTGCAGTCGTCC

MIRU 2

TGGACTTGCAGCAATGGACCAACT

MIRU 27

TCGAAAGCCTCTGCGTGCCAGTAA

GCGATGTGAGCGTGCCACTCAA

0.66

MIRU 16

TCGGTGATCGGGTCCAGTCCAAGTA

CCCGTCGTGCAGCCCTGGTAC

0.66

Qub 11b

CGTAAGGGGGATGCGGGAAATAGG

CGAAGTGAATGGTGGTGGCAT

1

MIRU 23

CAGCGAAACGAACTGTGCTATCAC

CGTGTCCGAGCAGAAAAGGGTAT

0.66

Mtub 30

AGTCACCTTTCCTACCACTCGTAAC

ATTAGTAGGGCACTAGCACCTCAAG

0.66

CTGATTGGCTTCATACGGCTTTA

GTGCCGACGTGGTCTTGAT

0.66

MIRU31

9

10

12 215

MIRU 24

CGACCAAGATGTGCAGGAATACAT

GGGCGAGTTGAGCTCACAGAA

0.66

MIRU 26

CCCGCCTTCGAAACGTCGCT

TGGACATAGGCGACCAGGCGAATA

0.66

Mtub 29

AACCCATGTCAGCCAGGTTA

ATGATGGCACACCGAAGAAC

1

MIRU 40

GGGTTGCTGGATGACAACGTGT

GGGTGATCTCGGCGAAATCAGATA

0.66

Mtub 34

GCAGATAACCCGCAGGAATA

GGAGAGGATACGTGGATTTGAG

1

GTCCAGGTTGCAAGAGATGG

GGCATCCTCAACAACGGTAG

0.66

Qub 4156

TGACCACGGATTGCTCTAGT

GCCGGCGTCCATGTT

1

Mtub 21

AGATCCCAGTTGTCGTCGTC

CAACATCGCCTGGTTCTGTA

0.66

Mtub 39

AATCACGGTAACTTGGGTTGTTT

GATGCATGTTCGACCCGTAG

1

Mtub 04

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(ETR-E)

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(µM)

MIRU 10

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Sequence of R-primer

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Conc.

Sequence of F-primer

PCR Locus name

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Abbreviations: F=Forward. R=Reverse. Conc.= Final concentration of primers in PCR

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reactions.

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Repeat Size

2165 4348 2461 4052 0577 2059

75bp 53bp 57bp 111bp 58bp 77bp

0580

77bp

(253) 330

0960 0154 3006 2163 1644 2401 2531 2687

53bp 53bp 53bp 69bp 53bp 58bp 53bp 53bp

[535] (455) (551) [136] (618*) 261 [607*] 447

(588) 508 (604) 205 671* 319 (660) 500

3192

53bp

(545)

598

2347 2996 3171 0802 0424 4156 1955 3690

57bp 51pb 54bp 54bp 51bp 59bp 57pb 58bp

[179] (236) [460*] (511) 562 (171) 225 407 461 (177) 218 269 [563] 622 681 149 206 283 341

[172] 514*

3

4

5

6

7

8

9

10

11

322 646 235 375 230 591

397 699* 292 486 288 [668]

472 (752) (349) 597 346

(547) [805] (406) 708 404

(622) [858*] (463) 819

(697*)

(772)

(847*)

(922)

[997]

[520] 930

[577] 1041

[634] 1152

1263

1374

(462)

(520*)

(578)

407

(484)

561

(638*)

[715]

[792*]

[869]

[946]

643*

696

749

(802*)

(855)

[908]

[961]

[1013]

[1065]

(561) 657 274 724 (377) (713) (553)

[614] 709 343 777 435 766

[762] 412 (829*) (493) 819

481 (882) [551] 872

(550) [935] [609*] (925)

(619*) [988]

(688*) [1041]

[757] [1094]

[826*] [1147]

(978)

[1031]

[1084]

[1137]

[606]

[659]

[712]

651

704

757

(810)

(863)

(916)

293 613 279 515 320 740* 263 399

350 664 (333) 569 371 799 320 457

(407) 715 (387) 623 422 [858] 377 515

[464*] (766) [441] 677 (473) [917] (434) (573)

817 [495] (731) (524) [976] (491) (631)

(868)

(919)

[970]

[1021]

(785) (575) [1035] (548) (689)

[839] [626*]

[893] [677*]

[728*]

(605) (747)

(662) (805)

[719] [863]

$

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(247) (593) [121] 178 264

2

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ETR-A MIRU39 ETR-B Qub26 ETR-C MIRU20 MIRU04 (ETRD-1) MIRU10 MIRU02 MIRU27 Qub11b MIRU16 Mtub30 MIRU23 MIRU24 MIRU31 (ETR-E) Mtub29 MIRU26 Mtub34 MIRU40 Mtub04* Qub4156* Mtub21 Mtub39*

Position on Chr. in kb

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VNTR Markers

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Table 3- Correspondence table between amplicon sizes and number of repeats for each VNTR, presented for each duplex. 12

13

14

15

[890] [1037]

[1095]

1485

[895}

[1072]

[1123]

[776] [921]

[833] [979]

Note that amplicon sizes observed less than 6 times in our study (including published data) are presented under brackets; sizes found in less than 3 samples are presented under square brackets. H37Rv size (Paris strain) amplicons sizes are underlined. Ambiguous amplicon sizes (proximity between two amplicon sizes observed in the same duplex) are highlighted by an asterisk.

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Figures Figure 1 – Gel electrophoresis of two duplexes for 29 samples and controls. A. MIRU2-MIRU27. B. MIRU4(=ETRD)-MIRU10. Samples are in the same order for the

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two gels. – indicates negative control (water); + indicates positive control H37Rv; sm=size marker.

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Supplemental information 1: Correspondence table presented tested duplexes. Orange highlights couple for which one of the primer couple did not amplify.

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Supplemental information 2: Genotypes of the 208 samples successfully analyzed (at

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least 22 VNTR data).

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