- Email: [email protected]
Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates
Accepted Manuscript Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates
Junji Seto, Takayuki Wada, Yu Suzuki, Tatsuya Ikeda, Katsumi Mizuta, Satoshi Mitarai, Tadayuki Ahiko PII: DOI: Reference:
S0167-7012(17)30095-7 doi: 10.1016/j.mimet.2017.04.008 MIMET 5151
To appear in:
Journal of Microbiological Methods
Received date: Revised date: Accepted date:
13 March 2017 11 April 2017 20 April 2017
Please cite this article as: Junji Seto, Takayuki Wada, Yu Suzuki, Tatsuya Ikeda, Katsumi Mizuta, Satoshi Mitarai, Tadayuki Ahiko , Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mimet(2017), doi: 10.1016/j.mimet.2017.04.008
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.
ACCEPTED MANUSCRIPT
REVISED Short Communication: Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates Junji Seto1*, Takayuki Wada2, Yu Suzuki1, Tatsuya Ikeda1, Katsumi Mizuta1, Satoshi Mitarai3, and Tadayuki Ahiko1 Department of Microbiology, Yamagata Prefectural Institute of Public Health, 1-6-6
Department of International Health, Institute of Tropical Medicine, Nagasaki
SC
2
RI
Toka-machi, Yamagata-shi, Yamagata 990-0031, Japan
PT
1
University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
Department of Mycobacterium Reference and Research, the Research Institute of
NU
3
MA
Tuberculosis, Japan Anti-tuberculosis Association, 3-1-24 Matsuyama, Kiyose,
D
Tokyo 204-8533, Japan
PT E
* Corresponding author: Junji Seto, Department of Microbiology, Yamagata Prefectural Institute of Public Health, Tokamachi 1-6-6, Yamagata, Yamagata 990-0031 Japan Tel.:
CE
+81-23-627-1373, Fax: +81-23-641-7486. E-mail:[email protected]
AC
Running title: Convenient VNTR typing for M. tuberculosis
1
ACCEPTED MANUSCRIPT
Summary Variable-number tandem-repeat typing for Mycobacterium tuberculosis clinical isolates contributes to evidence-based tuberculosis control. However, cumbersome PCR procedures for the typing have disturbed routine analyses. We proposed a convenient
PT
PCR method for the typing using a PCR master mix that provides rapidity and
SC
RI
long-term stability of the frozen PCR cocktail.
AC
CE
PT E
D
MA
rapidity, SapphireAmp Fast Master Mix
NU
Key words: frozen PCR cocktail, high labor effectiveness, human error reduction,
2
ACCEPTED MANUSCRIPT
Main text To detect tuberculosis infection with the same Mycobacterium tuberculosis (Mtb) isolate, variable-number tandem-repeat (VNTR) typing is used worldwide (Anderson et al., 2014; Munang et al., 2015; Seto et al., 2017; Wang et al., 2014).
regions.
However,
the
PCR
procedures
are
cumbersome
and
RI
minisatellite
PT
VNTR typing is a useful genotyping method based on PCR targeting multiple
SC
time-consuming. The PCR procedures have been well established and routinely fixed, however improvement for more rapid and easy-to-use protocols have not been
NU
considered yet.
MA
We used two PCR methods to examine 24Beijing-VNTR typing, including 24-loci optimized for Beijing family Mtb isolates (Iwamoto et al., 2012). A method
D
using SapphireAmp Fast Master Mix (SA method: Takara Bio Inc.) was selected for its
PT E
rapidity and cost-efficiency. Another method using Ex-Taq hot start version (Ex-HS method: Takara Bio Inc.) is the gold standard of VNTR-typing for Mtb in Japan. As template, 100 L of solution from 300 L of DW containing Mtb clinical isolates of
CE
quarter biomass-loopful (3 mm diameter) from Ogawa medium or 100 L of MGIT
AC
(Becton, Dickinson and Co.) culturing Mtb was heated to 100°C for 10 min. Then, the supernatants were prepared. The 10 L of PCR cocktail for the SA method contained 5L of 2master mix, 0.5 M of each forward/reverse primer (Maeda et al., 2008), and 0.5 L of template DNA. The PCR condition was at 94°C for 2 min; 40 cycles of 98°C for 5 s, 62°C for 5 s, and 72°C for 50 s; with final extension at 72°C for 1 min. In the Ex-HS method, we modified the method of an earlier study (Murase et al., 2008). We halved the total volume of PCR cocktail from 20 to 10 L and applied 0.5 L of 3
ACCEPTED MANUSCRIPT
template DNA. The PCR condition of the Ex-HS method was 94°C for 5 min, then 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 3 min, with final extension at 72°C for 7 min. All PCRs were performed using PCR Thermal Cycler Fast (Takara Bio Inc.). A microchip electrophoresis system (MCE-202; Shimadzu Corp.), an automated
PT
machine having high resolution and high reproducibility, was used to measure the PCR
RI
product size and concentration.
SC
The preliminary examination confirmed the concordance of 24Beijing-VNTR profiles between the SA and Ex-HS methods in four Mtb clinical isolates (data not
NU
shown). The PCR runtimes of SA and Ex-HS methods were 66 and 171 min,
MA
respectively. The reagents cost of the SA method (5.0 USD per a 24Beijing-VNTR analysis) was about half of the Ex-HS method (9.7 USD). Moreover, the accuracy of the
D
SA method was ensured by internal quality controls and an external quality assessment
lowers analysis costs.
PT E
(data not shown). Results indicate that the SA method raises labor effectiveness and
CE
To compare reactivities of the SA and Ex-HS methods in 24Beijing-VNTR typing, we measured the average concentration of PCR products in each locus among
AC
representative Mtb clinical isolates. We cultured eight Mtb clinical isolates with various VNTR profiles (Table S1) on Ogawa medium at 37°C for 30 days. After preparing the template DNA from the cultures, we performed the VNTR typing using the SA and Ex-HS methods. As to other conditions, we did not test in this study. The overall reactivity (i.e., average concentration of 192 PCR product) of the SA method was 42.4% compared to that of the Ex-HS method. The SA method amplified a sufficient amount of PCR products for analyzing the VNTR pattern in electrophoresis (i.e., > 1 4
ACCEPTED MANUSCRIPT
ng/L). However, almost all loci in the SA method showed lower concentration than that of the Ex-HS method (Fig. 1). On the basis of sufficient performance of the SA method, we determined the standard operating procedure for 24Beijing-VNTR typing of Mtb clinical isolates; the SA
PT
method was first performed for isolates. Then the Ex-HS method was fulfilled in the
RI
loci of the SA method that PCR product was absent or electrophoresis showed multiple
SC
bands. During April 2016 – January 2017, we typed for 58 Mtb clinical isolates (42 isolates on Ogawa medium and 16 isolates in MGIT). Consequently, we applied the
NU
Ex-HS method only to 28 (2.0%) of 1392 PCR samples (data not shown). Fourteen
MA
samples of the Ex-HS method showed the same results as those obtained from the SA method because a part of Mtb clinical isolates lacked loci of QUB-11a, JATA05, and
D
ETR-A in Rv1917c gene (Seto et al., 2015), or sometimes showed multiple repeats (e.g.,
PT E
no. Jc4 in Table S1). The Ex-HS method recovered the results of the SA methods in 14 (1.0%) of the 1392 samples. The proportion of the templates from Ogawa medium
CE
(6/1008: 0.6%) was significantly lower (p=0.03, Fisher’s exact test) than that of templates from MGIT (8/384: 2.1%). Furthermore, the DNA concentration of the 42
AC
templates from Ogawa medium [median (interquartile range): 20.7 (11.1–29.0)] was significantly higher (p<0.001, Wilcoxon rank sum test) than that of the 16 templates from MGIT [2.4 (2.0–4.5)]. Results suggest that the SA method is applicable for almost all routine analyses. However, some isolates, especially templates with low DNA concentration, might require re-examination using the Ex-HS method to obtain accurate results. Finally, we examined the storage stability of PCR cocktails to improve 5
ACCEPTED MANUSCRIPT
cumbersome VNTR typing. For immediate use, we prepared frozen PCR cocktails without template for 24Beijing-VNTR typing, which were pre-aliquoted into 0.2 ml PCR tubes and stored at -30°C. We thawed the cocktails for 0–12 weeks, and conducted VNTR typing using a common Mtb template DNA pre-aliquoted and stored at -30°C.
PT
The frozen PCR cocktails were amplified with stability for 12 weeks using the
RI
respective methods (Fig. 2). The frozen cocktails are a solution to cumbersome PCR
SC
procedures for VNTR typing. Furthermore, bulk preparation of the same PCR cocktail might improve working efficiency, reduce human error, and ensure uniform typing
NU
quality.
MA
In conclusion, we proposed a new PCR method which can shorten the runtime for VNTR typing of Mtb clinical isolates (Table). Furthermore, the frozen PCR
D
cocktails of the method showed stable amplification up to 12 weeks. Those results
PT E
indicate that the SA method will reduce the workload. This work was supported by the Research Program on Emerging and
CE
Re-emerging Infectious Diseases (15fk0108017h0002 and 15fk0108004h001) from Japan Agency for Medical Research and development.
AC
CONFLICT OF INTEREST
All authors declare that they have no conflict of interest related to this study.
6
ACCEPTED MANUSCRIPT
References Anderson, L.F., Tamne, S., Brown, T. et al., 2014. Transmission of multidrug-resistant tuberculosis in the UK: a cross-sectional molecular and epidemiological study of clustering
and
contact
tracing.
Lancet
Infect.
14,
406–415.
PT
http://dx.doi.org/10.1016/S1473-3099(14)70022-2
Dis.
RI
Iwamoto, T., Grandjean, L., Arikawa, K. et al. 2012. Genetic diversity and transmission
SC
characteristics of Beijing family strains of Mycobacterium tuberculosis in Peru. PLoS One. 7:e49651. http://dx.doi.org/10.1371/journal.pone.0049651
NU
Maeda, S., Murase, Y., Mitarai, S. et al., 2008. Rapid, simple genotyping method by the
MA
variable number of tandem repeats (VNTR) for Mycobacterium tuberculosis isolates in Japan–analytical procedure of JATA (12)-VNTR. Kekkaku. 83, 673–678 (in
D
Japanese).
PT E
Munang, M.L., Browne, C., Khanom, S. et al., 2015. Tuberculosis microepidemics among dispersed migrants, Birmingham, UK, 2004-2013. Emerg. Infect. Dis. 21,
CE
524–527. http://dx.doi.org/10.3201/eid2103.140209 Murase, Y., Mitarai, S., Sugawara, I. et al., 2008. Promising loci of variable number of
AC
tandem repeats for typing Beijing family Mycobacterium tuberculosis. J. Med. Microbiol. 57, 873–880. http://dx.doi.org/10.1099/jmm.0.47564-0 Seto, J., Wada, T., Iwamoto, T. et al., 2015. Phylogenetic assignment of Mycobacterium tuberculosis Beijing clinical isolates in Japan by maximum a posteriori estimation. Infect. Genet. Evol. 35, 82–88. http://dx.doi.org/10.1016/j.meegid.2015.07.029 Seto, J., Wada, T., Suzuki, Y. et al., 2017. Mycobacterium tuberculosis Transmission among Elderly Persons, Yamagata Prefecture, Japan, 2009–2015. Emerg. Infect. Dis. 7
ACCEPTED MANUSCRIPT
23, 448-455, 2017. http://dx.doi.org/10.3201/eid2303.161571 Wang, W., Mathema, B., Hu, Y. et al., 2014, Role of casual contacts in the recent transmission of tuberculosis in settings with high disease burden. Clin. Microbiol.
AC
CE
PT E
D
MA
NU
SC
RI
PT
Infect. 20, 1140–1145. http://dx.doi.org/10.1111/1469-0691.12726
8
ACCEPTED MANUSCRIPT
Table The condition of two PCR methods for variable-number tandem-repeat typing of Mycobacterium tuberculosis isolates used in this study SapphireAmp Fast Master Ex-Taq hot start version methodb
1 cycle
1 cycle
PT
PCR condition
Mix methoda
94°C for 5 min
RI
94°C for 2 min
35 cycles
SC
40 cycles
62°C for 5 s
MA
72°C for 50 s
94°C for 30 s
NU
98°C for 5 s
1 cycle
66 min
Proposed in this study
b
Murase et al., 2008
c
Modified the original PCR condition
AC
CE
a
72°C for 3 min 1 cycle 72°C for 7 min 171 min
PT E
Total run length
D
72°C for 1 min
62°Cc for 30 s
9
ACCEPTED MANUSCRIPT
Figure legends Fig. 1. Average concentration (± S.E.) of PCR products in each locus of 24Beijing-VNTR typing using templates of eight Mycobacterium tuberculosis clinical isolates. The examination was performed using SapphireAmp Fast Master Mix (SA) and Ex-Taq hot
PT
start version (Ex-HS) methods. The SA method amplified a sufficient amount of PCR
RI
products for analyzing the VNTR pattern. However, the concentration of each locus
SC
with the exception of J03 in the SA method was low against the Ex-HS method. Fig. 2. Fluctuation of the PCR efficiency of frozen cocktails in SapphireAmp Fast
NU
Master Mix (SA) and Ex-Taq hot start version (Ex-HS) methods. Average concentration
MA
(± S.E.) of 24 PCR products in 24Beijing-VNTR typing, using a common template from an Mtb clinical isolate, are shown temporally. The frozen PCR cocktails were amplified
AC
CE
PT E
D
with stability during 12 weeks in common to the two methods.
10
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Figure 1
11
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Figure 2
12
ACCEPTED MANUSCRIPT
Highlights Variable-number tandem-repeat typing for Mycobacterium tuberculosis is useful.
PCR procedures for the typing are cumbersome and take a long time.
A convenient PCR method for the typing was examined in this study.
The method is swift with long-term stability of freezing.
AC
CE
PT E
D
MA
NU
SC
RI
PT
13
Junji Seto, Takayuki Wada, Yu Suzuki, Tatsuya Ikeda, Katsumi Mizuta, Satoshi Mitarai, Tadayuki Ahiko PII: DOI: Reference:
S0167-7012(17)30095-7 doi: 10.1016/j.mimet.2017.04.008 MIMET 5151
To appear in:
Journal of Microbiological Methods
Received date: Revised date: Accepted date:
13 March 2017 11 April 2017 20 April 2017
Please cite this article as: Junji Seto, Takayuki Wada, Yu Suzuki, Tatsuya Ikeda, Katsumi Mizuta, Satoshi Mitarai, Tadayuki Ahiko , Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mimet(2017), doi: 10.1016/j.mimet.2017.04.008
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.
ACCEPTED MANUSCRIPT
REVISED Short Communication: Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates Junji Seto1*, Takayuki Wada2, Yu Suzuki1, Tatsuya Ikeda1, Katsumi Mizuta1, Satoshi Mitarai3, and Tadayuki Ahiko1 Department of Microbiology, Yamagata Prefectural Institute of Public Health, 1-6-6
Department of International Health, Institute of Tropical Medicine, Nagasaki
SC
2
RI
Toka-machi, Yamagata-shi, Yamagata 990-0031, Japan
PT
1
University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
Department of Mycobacterium Reference and Research, the Research Institute of
NU
3
MA
Tuberculosis, Japan Anti-tuberculosis Association, 3-1-24 Matsuyama, Kiyose,
D
Tokyo 204-8533, Japan
PT E
* Corresponding author: Junji Seto, Department of Microbiology, Yamagata Prefectural Institute of Public Health, Tokamachi 1-6-6, Yamagata, Yamagata 990-0031 Japan Tel.:
CE
+81-23-627-1373, Fax: +81-23-641-7486. E-mail:[email protected]
AC
Running title: Convenient VNTR typing for M. tuberculosis
1
ACCEPTED MANUSCRIPT
Summary Variable-number tandem-repeat typing for Mycobacterium tuberculosis clinical isolates contributes to evidence-based tuberculosis control. However, cumbersome PCR procedures for the typing have disturbed routine analyses. We proposed a convenient
PT
PCR method for the typing using a PCR master mix that provides rapidity and
SC
RI
long-term stability of the frozen PCR cocktail.
AC
CE
PT E
D
MA
rapidity, SapphireAmp Fast Master Mix
NU
Key words: frozen PCR cocktail, high labor effectiveness, human error reduction,
2
ACCEPTED MANUSCRIPT
Main text To detect tuberculosis infection with the same Mycobacterium tuberculosis (Mtb) isolate, variable-number tandem-repeat (VNTR) typing is used worldwide (Anderson et al., 2014; Munang et al., 2015; Seto et al., 2017; Wang et al., 2014).
regions.
However,
the
PCR
procedures
are
cumbersome
and
RI
minisatellite
PT
VNTR typing is a useful genotyping method based on PCR targeting multiple
SC
time-consuming. The PCR procedures have been well established and routinely fixed, however improvement for more rapid and easy-to-use protocols have not been
NU
considered yet.
MA
We used two PCR methods to examine 24Beijing-VNTR typing, including 24-loci optimized for Beijing family Mtb isolates (Iwamoto et al., 2012). A method
D
using SapphireAmp Fast Master Mix (SA method: Takara Bio Inc.) was selected for its
PT E
rapidity and cost-efficiency. Another method using Ex-Taq hot start version (Ex-HS method: Takara Bio Inc.) is the gold standard of VNTR-typing for Mtb in Japan. As template, 100 L of solution from 300 L of DW containing Mtb clinical isolates of
CE
quarter biomass-loopful (3 mm diameter) from Ogawa medium or 100 L of MGIT
AC
(Becton, Dickinson and Co.) culturing Mtb was heated to 100°C for 10 min. Then, the supernatants were prepared. The 10 L of PCR cocktail for the SA method contained 5L of 2master mix, 0.5 M of each forward/reverse primer (Maeda et al., 2008), and 0.5 L of template DNA. The PCR condition was at 94°C for 2 min; 40 cycles of 98°C for 5 s, 62°C for 5 s, and 72°C for 50 s; with final extension at 72°C for 1 min. In the Ex-HS method, we modified the method of an earlier study (Murase et al., 2008). We halved the total volume of PCR cocktail from 20 to 10 L and applied 0.5 L of 3
ACCEPTED MANUSCRIPT
template DNA. The PCR condition of the Ex-HS method was 94°C for 5 min, then 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 3 min, with final extension at 72°C for 7 min. All PCRs were performed using PCR Thermal Cycler Fast (Takara Bio Inc.). A microchip electrophoresis system (MCE-202; Shimadzu Corp.), an automated
PT
machine having high resolution and high reproducibility, was used to measure the PCR
RI
product size and concentration.
SC
The preliminary examination confirmed the concordance of 24Beijing-VNTR profiles between the SA and Ex-HS methods in four Mtb clinical isolates (data not
NU
shown). The PCR runtimes of SA and Ex-HS methods were 66 and 171 min,
MA
respectively. The reagents cost of the SA method (5.0 USD per a 24Beijing-VNTR analysis) was about half of the Ex-HS method (9.7 USD). Moreover, the accuracy of the
D
SA method was ensured by internal quality controls and an external quality assessment
lowers analysis costs.
PT E
(data not shown). Results indicate that the SA method raises labor effectiveness and
CE
To compare reactivities of the SA and Ex-HS methods in 24Beijing-VNTR typing, we measured the average concentration of PCR products in each locus among
AC
representative Mtb clinical isolates. We cultured eight Mtb clinical isolates with various VNTR profiles (Table S1) on Ogawa medium at 37°C for 30 days. After preparing the template DNA from the cultures, we performed the VNTR typing using the SA and Ex-HS methods. As to other conditions, we did not test in this study. The overall reactivity (i.e., average concentration of 192 PCR product) of the SA method was 42.4% compared to that of the Ex-HS method. The SA method amplified a sufficient amount of PCR products for analyzing the VNTR pattern in electrophoresis (i.e., > 1 4
ACCEPTED MANUSCRIPT
ng/L). However, almost all loci in the SA method showed lower concentration than that of the Ex-HS method (Fig. 1). On the basis of sufficient performance of the SA method, we determined the standard operating procedure for 24Beijing-VNTR typing of Mtb clinical isolates; the SA
PT
method was first performed for isolates. Then the Ex-HS method was fulfilled in the
RI
loci of the SA method that PCR product was absent or electrophoresis showed multiple
SC
bands. During April 2016 – January 2017, we typed for 58 Mtb clinical isolates (42 isolates on Ogawa medium and 16 isolates in MGIT). Consequently, we applied the
NU
Ex-HS method only to 28 (2.0%) of 1392 PCR samples (data not shown). Fourteen
MA
samples of the Ex-HS method showed the same results as those obtained from the SA method because a part of Mtb clinical isolates lacked loci of QUB-11a, JATA05, and
D
ETR-A in Rv1917c gene (Seto et al., 2015), or sometimes showed multiple repeats (e.g.,
PT E
no. Jc4 in Table S1). The Ex-HS method recovered the results of the SA methods in 14 (1.0%) of the 1392 samples. The proportion of the templates from Ogawa medium
CE
(6/1008: 0.6%) was significantly lower (p=0.03, Fisher’s exact test) than that of templates from MGIT (8/384: 2.1%). Furthermore, the DNA concentration of the 42
AC
templates from Ogawa medium [median (interquartile range): 20.7 (11.1–29.0)] was significantly higher (p<0.001, Wilcoxon rank sum test) than that of the 16 templates from MGIT [2.4 (2.0–4.5)]. Results suggest that the SA method is applicable for almost all routine analyses. However, some isolates, especially templates with low DNA concentration, might require re-examination using the Ex-HS method to obtain accurate results. Finally, we examined the storage stability of PCR cocktails to improve 5
ACCEPTED MANUSCRIPT
cumbersome VNTR typing. For immediate use, we prepared frozen PCR cocktails without template for 24Beijing-VNTR typing, which were pre-aliquoted into 0.2 ml PCR tubes and stored at -30°C. We thawed the cocktails for 0–12 weeks, and conducted VNTR typing using a common Mtb template DNA pre-aliquoted and stored at -30°C.
PT
The frozen PCR cocktails were amplified with stability for 12 weeks using the
RI
respective methods (Fig. 2). The frozen cocktails are a solution to cumbersome PCR
SC
procedures for VNTR typing. Furthermore, bulk preparation of the same PCR cocktail might improve working efficiency, reduce human error, and ensure uniform typing
NU
quality.
MA
In conclusion, we proposed a new PCR method which can shorten the runtime for VNTR typing of Mtb clinical isolates (Table). Furthermore, the frozen PCR
D
cocktails of the method showed stable amplification up to 12 weeks. Those results
PT E
indicate that the SA method will reduce the workload. This work was supported by the Research Program on Emerging and
CE
Re-emerging Infectious Diseases (15fk0108017h0002 and 15fk0108004h001) from Japan Agency for Medical Research and development.
AC
CONFLICT OF INTEREST
All authors declare that they have no conflict of interest related to this study.
6
ACCEPTED MANUSCRIPT
References Anderson, L.F., Tamne, S., Brown, T. et al., 2014. Transmission of multidrug-resistant tuberculosis in the UK: a cross-sectional molecular and epidemiological study of clustering
and
contact
tracing.
Lancet
Infect.
14,
406–415.
PT
http://dx.doi.org/10.1016/S1473-3099(14)70022-2
Dis.
RI
Iwamoto, T., Grandjean, L., Arikawa, K. et al. 2012. Genetic diversity and transmission
SC
characteristics of Beijing family strains of Mycobacterium tuberculosis in Peru. PLoS One. 7:e49651. http://dx.doi.org/10.1371/journal.pone.0049651
NU
Maeda, S., Murase, Y., Mitarai, S. et al., 2008. Rapid, simple genotyping method by the
MA
variable number of tandem repeats (VNTR) for Mycobacterium tuberculosis isolates in Japan–analytical procedure of JATA (12)-VNTR. Kekkaku. 83, 673–678 (in
D
Japanese).
PT E
Munang, M.L., Browne, C., Khanom, S. et al., 2015. Tuberculosis microepidemics among dispersed migrants, Birmingham, UK, 2004-2013. Emerg. Infect. Dis. 21,
CE
524–527. http://dx.doi.org/10.3201/eid2103.140209 Murase, Y., Mitarai, S., Sugawara, I. et al., 2008. Promising loci of variable number of
AC
tandem repeats for typing Beijing family Mycobacterium tuberculosis. J. Med. Microbiol. 57, 873–880. http://dx.doi.org/10.1099/jmm.0.47564-0 Seto, J., Wada, T., Iwamoto, T. et al., 2015. Phylogenetic assignment of Mycobacterium tuberculosis Beijing clinical isolates in Japan by maximum a posteriori estimation. Infect. Genet. Evol. 35, 82–88. http://dx.doi.org/10.1016/j.meegid.2015.07.029 Seto, J., Wada, T., Suzuki, Y. et al., 2017. Mycobacterium tuberculosis Transmission among Elderly Persons, Yamagata Prefecture, Japan, 2009–2015. Emerg. Infect. Dis. 7
ACCEPTED MANUSCRIPT
23, 448-455, 2017. http://dx.doi.org/10.3201/eid2303.161571 Wang, W., Mathema, B., Hu, Y. et al., 2014, Role of casual contacts in the recent transmission of tuberculosis in settings with high disease burden. Clin. Microbiol.
AC
CE
PT E
D
MA
NU
SC
RI
PT
Infect. 20, 1140–1145. http://dx.doi.org/10.1111/1469-0691.12726
8
ACCEPTED MANUSCRIPT
Table The condition of two PCR methods for variable-number tandem-repeat typing of Mycobacterium tuberculosis isolates used in this study SapphireAmp Fast Master Ex-Taq hot start version methodb
1 cycle
1 cycle
PT
PCR condition
Mix methoda
94°C for 5 min
RI
94°C for 2 min
35 cycles
SC
40 cycles
62°C for 5 s
MA
72°C for 50 s
94°C for 30 s
NU
98°C for 5 s
1 cycle
66 min
Proposed in this study
b
Murase et al., 2008
c
Modified the original PCR condition
AC
CE
a
72°C for 3 min 1 cycle 72°C for 7 min 171 min
PT E
Total run length
D
72°C for 1 min
62°Cc for 30 s
9
ACCEPTED MANUSCRIPT
Figure legends Fig. 1. Average concentration (± S.E.) of PCR products in each locus of 24Beijing-VNTR typing using templates of eight Mycobacterium tuberculosis clinical isolates. The examination was performed using SapphireAmp Fast Master Mix (SA) and Ex-Taq hot
PT
start version (Ex-HS) methods. The SA method amplified a sufficient amount of PCR
RI
products for analyzing the VNTR pattern. However, the concentration of each locus
SC
with the exception of J03 in the SA method was low against the Ex-HS method. Fig. 2. Fluctuation of the PCR efficiency of frozen cocktails in SapphireAmp Fast
NU
Master Mix (SA) and Ex-Taq hot start version (Ex-HS) methods. Average concentration
MA
(± S.E.) of 24 PCR products in 24Beijing-VNTR typing, using a common template from an Mtb clinical isolate, are shown temporally. The frozen PCR cocktails were amplified
AC
CE
PT E
D
with stability during 12 weeks in common to the two methods.
10
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Figure 1
11
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Figure 2
12
ACCEPTED MANUSCRIPT
Highlights Variable-number tandem-repeat typing for Mycobacterium tuberculosis is useful.
PCR procedures for the typing are cumbersome and take a long time.
A convenient PCR method for the typing was examined in this study.
The method is swift with long-term stability of freezing.
AC
CE
PT E
D
MA
NU
SC
RI
PT
13